bluuebunny/crossref_metadata_2025 huggingface dataset:
Clinical
Escherichia coli
Strains Carrying
stx
Genes:
stx
Variants and
stx
-Positive Virulence Profiles
Authors: Eklund Marjut , Leino Kirsikka , Siitonen Anja
Published: November 2002

Altogether, 173 Shiga toxin-producing
Escherichia coli
(STEC) serotype O157 (
n
= 111) and non-O157 (
n
= 62) isolates from 170 subjects were screened by PCR-restriction fragment length polymorphism for eight different
stx
genes. The results were compiled according to serotypes, phage types of O157, production of Stx toxin and enterohemolysin, and the presence of
eae
. The
stx
genes occurred in 11 combinations; the most common were
stx
2
with
stx
2c
(42%),
stx
2
alone (21%), and s
tx
1
alone (16%). Of the O157 strains, 64% carried
stx
2
with
stx
2c
versus 2% of the non-O157 strains (
P
< 0.001). In the non-O157 strains, the prevailing gene was s
tx
1
(99% versus 1% in O157 strains;
P
< 0.001). In addition, one strain (O Rough:H4:
stx
2c
) which has not previously been described as associated with hemolytic-uremic syndrome (HUS) was found. Ten
stx
-positive virulence profiles were responsible for 71% of all STEC infections. Of these profiles, five accounted for 71% of the 21 strains isolated from 20 patients with HUS or thrombotic thrombocytopenic purpura (TTP). The strains having the virulence profile that caused mainly HUS or TTP or bloody diarrhea produced Stx with titers of ≥1:128 (90%) more commonly than did other strains (51%;
P
< 0.001). These strains were also more commonly enterohemolytic (98% versus 68% for other strains;
P
< 0.001) and possessed the
eae
gene (100%) more commonly than did other strains (74%;
P
< 0.001). A particular virulence profile, O157:H7:PT2:
stx
2
:
stx
2c
:
eae
:Ehly, was significantly more frequently associated with HUS and bloody diarrhea than were other profiles (
P
= 0.02) and also caused the deaths of two children. In this study, the risk factors for severe symptoms were an age of <5 years and infection by the strain of O157:H7:PT2 mentioned above.
Evaluation of a New Rapid Test for the Combined Detection of Hepatitis B Virus Surface Antigen and Hepatitis B Virus e Antigen
Authors: Clement F. , Dewint P. , Leroux-Roels G.
Published: November 2002

There are about 350 million chronic hepatitis B virus (HBV) carriers worldwide. A proactive approach to the management of this disease is likely to reduce the morbidity and mortality caused by HBV. This study aimed to evaluate the diagnostic performance of a novel tool for discriminating between infected and noninfected subjects, the hepatitis B sAg/eAg test (Binax Inc., Portland, Maine). The test is designed to rapidly and accurately detect both the HBV surface antigen (HBsAg) and the HBV e antigen (HBeAg). A cohort of 942 subjects was tested. The serum clinical sensitivity of the hepatitis B sAg/eAg test was 99.75 and 96.37% for HBsAg and HBeAg, respectively. Serum clinical specificity was 99.32% for HBsAg and 98.99% for HBeAg. Analytical sensitivity was satisfactory for the purposes of population screening. Visual evaluation showed that the test signals were stable for at least 3 h after the recommended evaluation time. No interference or cross-reactivity was observed with known interfering substances and virologic markers. These results indicate that the hepatitis B sAg/eAg test is well suited to the accurate detection of HBV carriers. In addition to the good clinical specificity and sensitivity of this test, its stability and user-friendly design mean that a correct performance, even under field conditions, is highly likely. Consequently, the hepatitis B sAg/eAg test has the potential to identify subjects who require HBV vaccination (HBsAg
−
and HBeAg
−
) and HBV-infected individuals who might benefit most from antiviral therapy (HBsAg
+
and HBeAg
+
).
Use of Subtractive Hybridization To Identify a Diagnostic Probe for a Cystic Fibrosis Epidemic Strain of
Pseudomonas aeruginosa
Authors: Parsons Yasmin N. , Panagea Stavroula , Smart Catherine H. M. , Walshaw Martin J. , Hart C. Anthony , Winstanley Craig
Published: November 2002

A multiresistant strain of
Pseudomonas aeruginosa
is widespread among cystic fibrosis (CF) patients attending clinics in Liverpool, United Kingdom. Suppression subtractive hybridization was used to identify sequences present in the Liverpool CF epidemic strain but absent from strain PAO1. Using dot blot and PCR amplification assays, the prevalence of such sequences among a panel of CF isolates was determined. Several sequences were found only in the Liverpool epidemic strain. Some sequences were present in the Liverpool epidemic strain and in a minority of other isolates, including sequences with homology to genes implicated in O6 serotype and siderophore production. The Liverpool epidemic strain and 81% of nonepidemic isolates contained a sequence identified as part of the PAGI-1 genomic island. Other strains implicated in epidemic spread, which were from Manchester, United Kingdom, and Melbourne, Australia, were also screened. None of the sequences identified was present in the Manchester strain. However, one of two Melbourne strains contained some of the sequences found in the Liverpool epidemic strain. All isolates implicated in epidemic spread and 76% of sporadic isolates contained the
exoS
gene. A sequence present in all isolates of the Liverpool epidemic strain was used to develop a diagnostic PCR test for identification of the strain from colonies or directly from sputum samples.
Bartonella henselaeandBartonella elizabethaeas Potential Canine Pathogens
Authors: Mexas Angela M. , Hancock Susan I. , Breitschwerdt Edward B.
Published: November 2002

Bartonella henselaeorBartonella elizabethaeDNA from EDTA-anticoagulated blood samples obtained from four dogs was amplified and sequenced. The results showed thatB. elizabethaeshould be added to the list ofBartonellaspecies (i.e.,B. vinsoniisubsp.berkhoffii,B. henselae, andB. clarridgeiae) that are currently recognized as infectious agents in dogs. Furthermore, these results may have potential zoonotic implications, particularly if dogs can serve as a previously unrecognized reservoir forB. henselae. Although the clinical relevance of these observations remains to be determined, it is possible that molecular diagnostic techniques such as PCR may help to implicate a spectrum ofBartonellaspp. as a cause of or a cofactor in chronic canine and human diseases of poorly defined causation.
Analysis of the Pathogenetic Basis for Shedding and Transmission of Ovine Gamma Herpesvirus 2
Authors: Hüssy Daniela , Janett Fredi , Albini Sarah , Stäuber Norbert , Thun Rico , Ackermann Mathias
Published: November 2002

Ovine herpesvirus 2 (OvHV-2), a member of the viral subfamily
Gammaherpesvirinae
, shares numerous similarities with human herpesvirus 8 (HHV-8). Both viruses are apathogenic in their healthy original host, may cause lymphoprolipherative diseases, cannot routinely be propagated in cell culture, and may be sexually transmitted. However, the pathways of sexual transmission of these viruses, as well as the underlying pathogenetic dynamics, are not well understood. Organs from naturally OvHV-2-infected, as well as OvHV-2-free, sheep were quantitatively analyzed for OvHV-2 by the DNA amplification techniques. The dynamics of OvHV-2 multiplication and excretion were monitored after experimental infections and, most importantly, subsequent to vasectomy. The OvHV-2 DNA load in various tissues and internal organs was not merely reflecting the viral DNA load in the bloodstream, which suggested compartmentalization of OvHV-2. Moreover, OvHV-2 DNA was detected at several portals for virus shedding, i.e., the respiratory, alimentary, and urogenital tracts. Transient OvHV-2 excretion was detected in ejaculates of experimentally infected rams. Upon vasectomy, OvHV-2 DNA reappeared in the ejaculatory plasma, but the titers did not decline after reaching a peak. Spiking and fractionation experiments revealed an inhibitory activity, associated with the spermatozoa, which was able to suppress detection of viral DNA but which was no longer present in samples from vasectomized animals. Therefore, epidemiological studies on viruses that may be transmitted by the ejaculatory pathway and for whose tracing nucleic acid amplification methods are used, i.e., OvHV-2, HHV-8, and the human immunodeficiency virus, should include vasectomized males.
Endemic Carbapenem Resistance Associated with OXA-40 Carbapenemase among
Acinetobacter baumannii
Isolates from a Hospital in Northern Spain
Authors: Lopez-Otsoa F. , Gallego L. , Towner K. J. , Tysall L. , Woodford N. , Livermore D. M.
Published: November 2002

Eighty-two carbapenem-resistant isolates of
Acinetobacter baumannii
from a single hospital in Bilbao were typed into two major clusters and several subclusters. Disk synergy tests and PCR indicated the production of a zinc-independent OXA-class carbapenemase. Sequencing identified this enzyme, OXA-40, as a variant of the OXA-24-OXA-25-OXA-26 cluster.
Performance of the Microscopic Observation Drug Susceptibility Assay in Drug Susceptibility Testing for
Mycobacterium tuberculosis
Authors: Park Walter G. , Bishai William R. , Chaisson Richard E. , Dorman Susan E.
Published: November 2002

The drug susceptibility testing performance of a broth-based method with microscopic reading of bacillary growth, the microscopic observation drug susceptibility (MODS) assay, was compared to that of the reference 7H10 agar method of proportion by using 53 isolates of
Mycobacterium tuberculosis
from persons at risk for multidrug-resistant TB. For isoniazid (0.1 μg/ml) and rifampin (2.0 μg/ml), there was 100% agreement between MODS results read at day 11 and the reference method. Levels of agreement for ethambutol tested at 2.5 and 7.5 μg/ml were 70 and 58%, respectively. Levels of agreement for streptomycin tested at 2.0 and 6.0 μg/ml were 77 and 51%, respectively. For isoniazid and rifampin drug susceptibility testing, MODS is as accurate as and more rapid than the reference method.
Rotavirus Particles Can Survive Storage in Ambient Tropical Temperatures for More than 2 Months
Authors: Fischer Thea K. , Steinsland Hans , Valentiner-Branth Palle
Published: November 2002

Typing and in vitro cultivation of rotavirus-positive human stool samples stored unintentionally at ambient tropical temperatures for 2 1/2 months showed that rotavirus is stable and may still be infectious in vitro. This indicates that stool specimen collection for rotavirus studies can be performed in areas and settings where reliable cold storage is not available. The retained infectivity of rotavirus particles underscores the need for safe systems for disposal of feces, in particular in developing countries where rotavirus is a major cause of childhood mortality.
Overestimation of the Hepatitis C Virus RNA Content of Reference Preparations by the AMPLICOR HCV Monitor Test, Version 2.0
Authors: Pisani Giulio , Cristiano Karen , Wirz Maria , Bisso Guillermo M. , Gentili Giuliano
Published: November 2002

An evaluation of the AMPLICOR hepatitis C virus (HCV) monitor test, version 2.0 (Roche Diagnostics), was carried out to investigate whether this test overestimates the HCV RNA content of reference preparations. Satisfactory accuracy was observed when the World Health Organization HCV international standard was included in the assay and a modified formula was used to calculate the viral content.
Are There Naturally Occurring Pleomorphic Bacteria in the Blood of Healthy Humans?
Authors: McLaughlin Richard W. , Vali Hojatollah , Lau Peter C. K. , Palfree Roger G. E. , De Ciccio Angela , Sirois Marc , Ahmad Darakhshan , Villemur Richard , Desrosiers Marcel , Chan Eddie C. S.
Published: November 2002

Dark-field microscopy of blood from healthy individuals revealed the existence of pleomorphic microorganisms. These bacteria exhibited limited growth and susceptibility to antibiotics and could be detected by fluorescent in situ hybridization and flow cytometry. They were further characterized by analysis of their 16S rRNA and
gyrB
genes.
Characterization of a Toxigenic
Vibrio cholerae
O139 Strain Belonging to a New Ribotype and Isolated from a Diarrheal Patient
Authors: Bhanumathi R. , Sabeena F. , Isac Sree Renjini , Radhakutty G. , Singh D. V.
Published: November 2002

We characterized a
Vibrio cholerae
O139 strain isolated from a diarrheal patient admitted to Taluk Hospital, Cherthala, Alleppey, Kerala, India, on 9 June 2000. The
V. cholerae
O139 strain possesses the core of the CTX genetic element, colonization toxin-coregulated pilus, the adherence outer membrane protein, and the central regulatory protein encoded by
toxR
and produces cholera toxin (200 pg/ml). We provide molecular evidence showing that toxigenic
V. cholerae
O139 strain ALO95 belongs to a distinct genotype characterized by a unique ribotype designated B-VII and has a unique enterobacterial repetitive intergenic consensus sequence PCR fingerprint profile designated E-V.
Molecular Epidemiology of Tuberculosis in a Rural Area of High Prevalence in South India: Implications for Disease Control and Prevention
Authors: Narayanan Sujatha , Das Sulochana , Garg Renu , Hari Lalitha , Rao Vijay Bhaskara , Frieden Thomas R. , Narayanan P. R.
Published: November 2002

Molecular and conventional epidemiologic techniques were used to study the mechanisms and risk factors for tuberculosis transmission in a rural area with high prevalence in south India. Restriction fragment length polymorphism analysis with IS
6110
and direct repeat probes was performed with 378
Mycobacterium tuberculosis
isolates from patients. Forty-one percent of
M. tuberculosis
isolates harbored a single copy of IS
6110
. Of 378 patients, 236 had distinct strains; 142 (38%) shared a strain with other patients, indicating recent infection. Older patients, those detected by a house-to-house community survey, and those hospitalized in a sanatorium were more likely to have had a recent infection. These findings suggest that the majority of the tuberculosis cases in south India were due to reactivation; therefore, efforts to control tuberculosis should be sustained.
Effects of fatigue on the torque-velocity relation in muscle
Authors: Spendiff O , Longford N T , Winter E M
Published: November 2002

Background:The extent to which fatigue is related to the velocity of shortening is still not fully understood.

Objectives:To examine the effects of fatigue induced by maximal intensity exercise at different velocities on subsequent torque-velocity relations in muscle.

Methods:Ten men (mean (SD) age 25 (3) years; stature 1.78 (0.34) m; body mass 80.6 (14.5) kg) provided written informed consent and, over a five day period in a randomised manner, performed 16 fatiguing bouts of maximal intensity exercise on an isokinetic dynamometer each followed by one repetition maximum action. Concentric peak torque of the preferred leg was assessed at angular velocities of 0.52, 1.05, 2.09, and 3.14 rad/s. Mechanical work performed for all fatiguing bouts was held at 3050–3150 J. After a 45 second rest, a maximum effort was performed at one of the preset velocities. As a baseline, dependent variables of peak torque (N.m), angle of peak torque (rad), and mean torque (N.m) were measured at each velocity. An analysis of covariance was used to compare torque-velocity relations, and one factor, within subject analyses of variance with repeated measures investigated differences from baseline.

Results:Torque-velocity relations for all dependent variables did not differ (p>0.05). Similarly, angles at which peak torque occurred were not velocity dependent and did not differ from baseline and subsequent exercise measures (p>0.05). Generally, fatiguing exercise at low velocities led to reductions in peak and mean torque in subsequent exercise at higher velocities (p<0.05).

Conclusions:Torque-velocity relations fundamentally remain intact after fatigue induced at any velocity, although the magnitude changes. The results suggest that the greater decline in torque during subsequent exercise at high velocities could be due to greater exhaustion of fatigue sensitive type II fibres, whereas low velocity subsequent exercise is less affected because of the greater use of type I fibres.
Hyperextension trauma to the elbow: radiological and ultrasonographic evaluation in handball goalkeepers
Authors: Popovic N , Lemaire R
Published: November 2002

Objective:To detect elbow lesions produced by hyperextension in 30 elite handball goalkeepers.

Methods:Conventional radiographs, stress radiographs, and ultrasound examination of both elbows were used. As a control group, 30 male volunteers from the general population within the same age group with no history of elbow injury were used.

Results:Radiographic findings in the goalkeepers were osteophyte formation in 67%, loose bodies in 5.5%, and periarticular calcification in 5.5%. Significantly greater differences in medial joint space opening between stressed and unstressed elbows were measured in both elbows than in the control group. Ultrasonographic findings showed thickening of the medial collateral ligament in 50%, thickening of the tricipital tendon in 11%, and signs of ulnar neuritis in 22%. An intra-articular effusion was found in 66% and small loose bodies in 33%. No significant differences were found between the dominant and non-dominant elbows at radiological and ultrasound examination. The findings in the control group were normal.

Conclusions:The findings support the theory that repetitive hyperextension trauma to the elbow in handball goalkeepers results in pathological changes.
Flail arms in a parachutist: an unusual presentation of hereditary neuropathy with liability to pressure palsies
Authors: Marriott M , Macdonell R , McCrory P
Published: November 2002

Parachuting is a popular sport but carries with it an appreciable injury risk. The case is presented of severe arm weakness after a parachute jump in a susceptible young woman.
LONG-RANGE FRACTAL CORRELATIONS IN LITERARY CORPORA
Authors: MONTEMURRO MARCELO A. , PURY PEDRO A.
Published: November 2002

In this paper, we analyze the fractal structure of long human language records by mapping large samples of texts onto time series. The particular mapping set up in this work is inspired on linguistic basis in the sense that is retains the word as the fundamental unit of communication. The results confirm that beyond the short-range correlations resulting from syntactic rules acting at sentence level, long-range structures emerge in large written language samples that give rise to long-range correlations in the use of words.
ASYMPTOTICS FOR THE SPECTRAL AND WALK DIMENSION AS FRACTALS APPROACH EUCLIDEAN SPACE
Authors: HAMBLY B. M. , KUMAGAI T.
Published: November 2002

We discuss the behavior of the dynamic dimension exponents for families of fractals based on the Sierpinski gasket and carpet. As the length scale factor for the family tends to infinity, the lattice approximations to the fractals look more like the tetrahedral or cubic lattice in Euclidean space and the fractal dimension converges to that of the embedding space. However, in the Sierpinski gasket case, the spectral dimension converges to two for all dimensions. In two dimensions, we prove a conjecture made in the physics literature concerning the rate of convergence. On the other hand, for natural families of Sierpinski carpets, the spectral dimension converges to the dimension of the embedding Euclidean space. In general, we demonstrate that for both cases of finitely and infinitely ramified fractals, a variety of asymptotic values for the spectral dimension can be achieved.
Characterization of human blood dendritic cell subsets
Authors: MacDonald Kelli P. A. , Munster David J. , Clark Georgina J. , Dzionek Andrzej , Schmitz Juergen , Hart Derek N. J.
Published: November 2002

Dendritic cells (DCs) are key antigen-presenting cells for stimulating immune responses and they are now being investigated in clinical settings. Although defined as lineage-negative (Lin−) HLA-DR+ cells, significant heterogeneity in these preparations is apparent, particularly in regard to the inclusion or exclusion of CD14+, CD16+, and CD2+ cells. This study used flow cytometry and a panel of monoclonal antibodies (mAbs), including reagents from the 7th Leukocyte Differentiation Antigen Workshop, to define the cellular composition of 2 standardized peripheral blood mononuclear cell (PBMCs)–derived Lin− HLA-DR+preparations. Lin− cells were prepared from PBMCs by depletion with CD3, CD14, CD19, CD11b, and either CD16 or CD56 mAbs. Analysis of the CD16-replete preparations divided the Lin− HLA-DR+ population into 5 nonoverlapping subsets (mean ± 1 SD): CD123 (mean = 18.3% ± 9.7%), CD1b/c (18.6% ± 7.6%), CD16 (49.6% ± 8.5%), BDCA-3 (2.7% ± 1.4%), and CD34 (5.0% ± 2.4%). The 5 subsets had distinct phenotypes when compared with each other, monocytes, and monocyte-derived DCs (MoDCs). The CD85 family, C-type lectins, costimulatory molecules, and differentiation/activation molecules were also expressed differentially on the 5 Lin−HLA-DR+ subsets, monocytes, and MoDCs. The poor viability of CD123+ DCs in vitro was confirmed, but the CD16+ CD11c+ DC subset also survived poorly. Finally, the individual subsets used as stimulators in allogeneic mixed leukocyte reactions were ranked by their allostimulatory capacity as CD1b/c > CD16 > BDCA-3 > CD123 > CD34. These data provide an opportunity to standardize the DC populations used for future molecular, functional and possibly even therapeutic studies.
Third-generation, self-inactivating gp91phoxlentivector corrects the oxidase defect in NOD/SCID mouse–repopulating peripheral blood–mobilized CD34+ cells from patients with X-linked chronic granulomatous disease
Authors: Roesler Joachim , Brenner Sebastian , Bukovsky Anatoly A. , Whiting-Theobald Narda , Dull Thomas , Kelly Michael , Civin Curt I. , Malech Harry L.
Published: November 2002

HIV-1–derived lentivectors are promising for gene transfer into hematopoietic stem cells but require preclinical in vivo evaluation relevant to specific human diseases. Nonobese diabetic/severe combined immunodeficient (NOD/SCID) mice accept human hematopoietic stem cell grafts, providing a unique opportunity for in vivo evaluation of therapies targeting human hematopoietic diseases. We demonstrate for the first time that hematopoietic stem cells from patients with X-linked chronic granulomatous disease (X-CGD) give rise to X-CGD–phenotype neutrophils in the NOD/SCID model that can be corrected using VSV-G–pseudotyped, 3rd-generation, self-inactivating (SIN) lentivector encoding gp91phox. We transduced X-CGD patient-mobilized CD34+ peripheral blood stem cells (CD34+PBSCs) with lentivector–gp91phox or amphotropic oncoretrovirus MFGS–gp91phox and evaluated correction ex vivo and in vivo in NOD/SCID mice. Only lentivector transduced CD34+PBSCs under ex vivo conditions nonpermissive for cell division, but both vectors performed best under conditions permissive for proliferation (multiple growth factors). Under the latter conditions, lentivector and MFGS achieved significant ex vivo correction of X-CGD CD34+PBSCs (18% and 54% of cells expressing gp91phox, associated with 53% and 163% of normal superoxide production, respectively). However, lentivector, but not MFGS, achieved significant correction of human X-CGD neutrophils arising in vivo in NOD/SCID mice that underwent transplantation (20% and 2.4%, respectively). Thus, 3rd-generation SIN lentivector–gp91phox performs well as assessed in human X-CGD neutrophils differentiating in vivo, and our studies suggest that the NOD/SCID model is generally applicable for in vivo study of therapies evaluated in human blood cells expressing a specific disease phenotype.
Low NAD(P)H:quinone oxidoreductase activity is associated with increased risk of leukemia with MLL translocations in infants and children
Authors: Smith Martyn T. , Wang Yunxia , Skibola Christine F. , Slater Diana J. , Nigro Luca Lo , Nowell Peter C. , Lange Beverly J. , Felix Carolyn A.
Published: November 2002

An inactivating polymorphism at position 609 in the NAD(P)H:quinone oxidoreductase 1 gene (NQO1 C609T) is associated with an increased risk of adult leukemia. A small British study suggested thatNQO1 C609T was associated with an increased risk of infant leukemias with MLL translocations, especially infant acute lymphoblastic leukemia (ALL) with t(4;11). We explored NQO1 C609Tas a genetic risk factor in 39 pediatric de novo and 18 pediatric treatment-related leukemias with MLL translocations in the United States. Children with de novo B-lineage ALL withoutMLL translocations and a calculation of the expected genotype distribution in an ethnically matched population of disease-free subjects served as the comparison groups. Patients with de novo leukemias with MLL translocations were significantly more likely to be heterozygous at NQO1 C609T (odds ratio [OR] = 2.77, 95% confidence intervals [CI] 1.17-6.57;P = .02), and significantly more likely to have low/null NQO1 activity than patients with de novo B-lineage ALL withoutMLL translocations (OR = 2.47, 95% CI 1.08-5.68;P = .033). They were also significantly more likely to have low/null NQO1 activity than expected in an ethnically matched population of disease-free subjects (OR = 2.50,P = .02). Infants younger than 12 months old at diagnosis of leukemia with t(4;11) were most likely to have low/null NQO1 activity (OR > 10.0). Conversely, the distribution ofNQO1 genotypes among patients with treatment-related leukemias with MLL translocations was not statistically different than in the comparison groups. The inactivating NQO1polymorphism is associated with an increased risk of de novo leukemia with MLL translocations in infants and children.
IL-3–induced enhancement of retinoic acid receptor activity is mediated through Stat5, which physically associates with retinoic acid receptors in an IL-3–dependent manner
Authors: Si Jutong , Collins Steven J.
Published: November 2002

The regulation of hematopoiesis involves the interaction of specific hematopoietic cytokines with lineage-specific transcription factors, but little is known about how these cytokines might regulate the expression/activity of these different transcription factors. Here we identify the critical signal transduction pathways that mediate the interleukin 3 (IL-3)–induced enhancement of retinoic acid receptor (RAR) transcriptional activity that accompanies the IL-3–mediated commitment of the multipotent, stem cell factor (SCF)–dependent EML cell line to granulocyte/monocyte progenitors. We observe that the addition of IL-3 to EML cells induces activation of the phosphatidylinositol-3 kinase, mitogen-activated protein kinase, and Jak/Stat pathways and that Jak2 activation is the critical “proximal” mediator of the IL-3–induced enhancement of RAR activity. Constitutively active Stat5 constructs enhance both the transcriptional activity of RARs in EML cells and the commitment of these cells to granulocyte/monocyte progenitors, whereas dominant-negative Stat5 constructs inhibit this IL-3–induced enhancement of RAR transcriptional activity. We observe that the retinoic acid response element (RARE) used in our RA responsive reporter harbors overlapping Stat/RAR-binding sites. Moreover, coimmunoprecipitation studies indicate an interaction between Stat5 and RARs that is IL-3 dependent. Thus, Stat5 is an important mediator of the IL-3–induced enhancement of RAR transcriptional activity that accompanies the commitment of immature EML cells to the granulocyte/monocyte lineage. Cytokine-mediated physical and functional interactions between Stat5 and RARs may play critical roles in regulating different stages of hematopoiesis.
Fas-mediated apoptosis is important in regulating cell replication and death in trisomy 8 hematopoietic cells but not in cells with other cytogenetic abnormalities
Authors: Sloand Elaine M. , Kim Sonnie , Fuhrer Monika , Risitano Antonio M. , Nakamura Ryotaro , Maciejewski Jaroslaw P. , Barrett A. John , Young Neal S.
Published: November 2002

Increased apoptosis of hematopoietic progenitor cells has been implicated in the pathophysiology of cytopenias associated with myelodysplastic syndromes (MDSs), and inhibition by immunosuppression may account for the success of this treatment in some patients. We examined bone marrow and peripheral blood of 25 patients with chromosomal abnormalities associated with MDS (monosomy 7, trisomy 8, and 5q−) for evidence of apoptosis. When fresh bone marrow was examined, the number of apoptotic and Fas-expressing CD34 cells was increased in patients with trisomy 8, but decreased in monosomy 7, as compared with healthy control donor marrow. Fas expression was increased in the trisomy 8 cells and decreased in the monosomy 7 cells when compared with normal cells from the same patient. Trisomy 8 cells were more likely to express activated caspase-3 than were normal cells. For bone marrow cells cultured with Fas agonist or Fas antagonist, the percentage of cells with trisomy 8 was significantly decreased in most cases after Fas receptor triggering and increased by Fas ligand (Fas-L) antagonist (P < 0.01), suggesting increased Fas susceptibility of cells with trisomy 8. No such changes were seen in cultures of cells with 5q− or monosomy 7. Fas antagonist facilitated the expansion of cells with trisomy 8 only. Cells with trisomy 8 appear to be more susceptible to Fas-mediated apoptosis. Clinical data demonstrating the responsiveness of some patients with trisomy 8 to anti–thymocyte globulin (ATG) and cyclosporine (CsA) would favor an active role of the immune system in this syndrome.
Analysis of T-cell responses to Aspergillus fumigatus antigens in healthy individuals and patients with hematologic malignancies
Authors: Hebart Holger , Bollinger Claudia , Fisch Paul , Sarfati Jacqueline , Meisner Christoph , Baur Manuela , Loeffler Jürgen , Monod Michel , Latgé Jean-Paul , Einsele Hermann
Published: November 2002

Invasive aspergillosis has become a major cause of infection-related mortality in nonneutropenic patients after allogeneic stem cell transplantation (SCT). To assess the potential role ofAspergillus-specific T-cell responses for the successful control of invasive aspergillosis, lymphoproliferative responses toAspergillus fumigatus antigens were studied in healthy individuals, patients with evidence of invasive aspergillosis, and patients late after allogeneic SCT. In healthy individuals, a positive lymphoproliferative response was documented to cellular extracts of A fumigatus (14 of 16), the 88-kDa dipeptidylpeptidase (4 of 16), and the 90-kDa catalase (8 of 11). A predominant release of interferon γ (IFN-γ) in culture supernatants on stimulation with A fumigatus antigens was demonstrated in 13 of 17 healthy individuals, indicating a TH1 response. In patients with clinical evidence of invasive aspergillosis, a favorable response to antifungal therapy was found to correlate with a higher IFN-γ/interleukin 10 (IL-10) ratio in culture supernatants (n = 7; median ratio, IFN-γ/IL-10 = 1.0; range, 0.09-24.8) compared to 10 patients with progressive or stable disease (median ratio, IFN-γ/IL-10 = 0.1; range, 0.002-2.1; P = .04). Steroid treatment was found to suppressAspergillus-specific lymphoproliferation (P = .037) and release of IFN-γ in culture supernatants (P = .017). In contrast to cytomegalovirus- and tetanus toxoid–specific T-cell responses, Aspergillus-specific T-cell reconstitution late after allogeneic SCT was characterized by low stimulation indices and a low IFN-γ/IL-10 ratio. In addition, phosphoantigen-reactive Vγ9/Vδ2 T-cell clones from healthy individuals were found to produce significant amounts of tumor necrosis factor in response to A fumigatus antigens. In conclusion, these results further support the hypothesis that T cells contribute to the host defense against A fumigatus.
Ex vivo culture with human brain endothelial cells increases the SCID-repopulating capacity of adult human bone marrow
Authors: Chute John P. , Saini Abha A. , Chute Dennis J. , Wells Mark R. , Clark William B. , Harlan David M. , Park Jenny , Stull Margaret K. , Civin Curt , Davis Thomas A.
Published: November 2002

Adult human bone marrow (ABM) is an important source of hematopoietic stem cells for transplantation in the treatment of malignant and nonmalignant diseases. However, in contrast to the recent progress that has been achieved with umbilical cord blood, methods to expand ABM stem cells for therapeutic applications have been disappointing. In this study, we describe a novel culture method that uses human brain endothelial cells (HUBECs) and that supports the quantitative expansion of the most primitive measurable cell within the adult bone marrow compartment, the nonobese diabetic/severe combined immunodeficient (NOD/SCID) repopulating cell (SRC). Coculture of human ABM CD34+ cells with brain endothelial cells for 7 days supported a 5.4-fold increase in CD34+ cells, induced more than 95% of the CD34+CD38− subset to enter cell division, and produced progeny that engrafted NOD/SCID mice at significantly higher rates than fresh ABM CD34+ cells. Using a limiting dilution analysis, we found the frequency of SRCs within fresh ABM CD34+ cells to be 1 in 9.9 × 105 cells. Following HUBEC culture, the estimated frequency of SRCs increased to 1 in 2.4 × 105cells. All mice that received transplants of HUBEC-cultured cells showed B-lymphoid and myeloid differentiation, indicating that a primitive hematopoietic cell was preserved during culture. Noncontact HUBEC cultures also maintained SRCs at a level comparable to contact HUBEC cultures, suggesting that cell-to-cell contact was not required. These data demonstrate that human brain endothelial cells possess a unique hematopoietic activity that increases the repopulating capacity of adult human bone marrow.
Invasive aspergillosis in allogeneic stem cell transplant recipients: changes in epidemiology and risk factors
Authors: Marr Kieren A. , Carter Rachel A. , Boeckh Michael , Martin Paul , Corey Lawrence
Published: November 2002

The incidence of postengraftment invasive aspergillosis (IA) in hematopoietic stem cell transplant (HSCT) recipients increased during the 1990s. We determined risks for IA and outcomes among 1682 patients who received HSCTs between January 1993 and December 1998. Risk factors included host variables (age, underlying disease), transplant variables (stem cell source), and late complications (acute and chronic graft-versus-host disease [GVHD], receipt of corticosteroids, secondary neutropenia, cytomegalovirus [CMV] disease, and respiratory virus infection). We identified risk factors associated with IA early after transplantation (≤ 40 days) and after engraftment (41-180 days). Older patient age was associated with an increased risk during both periods. Chronic myelogenous leukemia (CML) in chronic phase was associated with low risk for early IA compared with other hematologic malignancies, aplastic anemia, and myelodysplastic syndrome. Multiple myeloma was associated with an increased risk for postengraftment IA. Use of human leukocyte antigen (HLA)–matched related (MR) peripheral blood stem cells conferred protection against early IA compared with use of MR bone marrow, but use of cord blood increased the risk of IA early after transplantation. Factors that increased risks for IA after engraftment included receipt of T cell–depleted or CD34-selected stem cell products, receipt of corticosteroids, neutropenia, lymphopenia, GVHD, CMV disease, and respiratory virus infections. Very late IA (> 6 months after transplantation) was associated with chronic GVHD and CMV disease. These results emphasize the postengraftment timing of IA; risk factor analyses verify previously recognized risk factors (GVHD, receipt of corticosteroids, and neutropenia) and uncover the roles of lymphopenia and viral infections in increasing the incidence of postengraftment IA in the 1990s.
Human CD5 promotes B-cell survival through stimulation of autocrine IL-10 production
Authors: Gary-Gouy Hélène , Harriague Julie , Bismuth Georges , Platzer Cornelia , Schmitt Christian , Dalloul Ali H.
Published: November 2002

CD5 is a negative regulator of B-cell receptor (BCR) signaling that is up-regulated after BCR stimulation and likely contributes to B-cell tolerance in vivo. However, CD5 is constitutively expressed on the B-1 subset of B cells. Contrary to CD5− B-2 B cells, B-1 B cells are long-lived because of autocrine interleukin-10 (IL-10) production through unknown mechanisms. We demonstrate herein a direct relationship between CD5 expression and IL-10 production. Human peripheral blood CD5+ B cells produce more IL-10 than CD5− B cells after BCR activation. Introducing CD5 into CD5− B cells induces the production of IL-10 by activating its promoter and the synthesis of its mRNA. The cytoplasmic domain of CD5 is sufficient for this process. CD5 also protects normal human B cells from apoptosis after BCR stimulation while reducing the BCR-induced Ca2+ response. We conclude that CD5 supports the survival of B cells by stimulating IL-10 production and by concurrently exerting negative feedback on BCR-induced signaling events that can promote cell death.
Congenital afibrinogenemia: first identification of splicing mutations in the fibrinogen Bβ-chain gene causing activation of cryptic splice sites
Authors: Spena Silvia , Duga Stefano , Asselta Rosanna , Malcovati Massimo , Peyvandi Flora , Tenchini Maria Luisa
Published: November 2002

Congenital afibrinogenemia is a rare inherited coagulopathy, characterized by very low or unmeasurable plasma levels of immunoreactive fibrinogen. So far, 25 mutations have been identified in afibrinogenemia, 17 in the Aα, 6 in the γ, and only 2 in the Bβ fibrinogen–chain genes. Here, 2 afibrinogenemic probands, showing undetectable levels of functional fibrinogen, were screened for causative mutations at the genomic level. Sequence analysis of the 3 fibrinogen genes disclosed 2 novel homozygous mutations in introns 6 and 7 of the Bβ-chain gene (IVS6 + 13C > T and IVS7 + 1G > T), representing the first Bβ-chain gene splicing mutations described in afibrinogenemia. The IVS6 + 13C > T mutation predicts the creation of a donor splice site in intron 6, whereas the IVS7 + 1G > T mutation causes the disappearance of the invariant GT dinucleotide of intron 7 donor splice site. To analyze the effect of these mutations, expression plasmids containing Bβ-chain minigene constructs, either wild-type or mutant, were transfected in HeLa cells. Assessed by semiquantitative analysis of reverse transcriptase–polymerase chain reaction products, the IVS7 + 1G > T mutation resulted in multiple aberrant splicings, while the IVS6 + 13C > T mutation resulted in activation of a new splice site 11 nucleotides downstream of the physiologic one. Both mutations are predicted to determine protein truncations, supporting the importance of the C-terminal domain of the Bβ chain for fibrinogen assembly and secretion.
Specific involvement of caspases in the differentiation of monocytes into macrophages
Authors: Sordet Olivier , Rébé Cédric , Plenchette Stéphanie , Zermati Yaël , Hermine Olivier , Vainchenker William , Garrido Carmen , Solary Eric , Dubrez-Daloz Laurence
Published: November 2002

Caspases are cysteine proteases involved in apoptosis and cytokine maturation. In erythroblasts, keratinocytes, and lens epithelial cells undergoing differentiation, enucleation has been regarded as a caspase-mediated incomplete apoptotic process. Here, we show that several caspases are activated in human peripheral blood monocytes whose differentiation into macrophages is induced by macrophage colony-stimulating factor (M-CSF). This activation is not associated with cell death and cannot be detected in monocytes undergoing dendritic cell differentiation in the presence of interleukin-4 (IL-4) and granulocyte-macrophage colony-stimulating factor (GM-CSF). The mechanisms and consequences of caspase activation were further studied in U937 human monocytic cells undergoing phorbol ester–induced differentiation into macrophages. Differentiation-associated caspase activation involves the release of cytochrome c from the mitochondria and leads to the cleavage of the protein acinus while the poly(ADP-ribose)polymerase remains uncleaved. Inhibition of caspases by either exposure to the broad-spectrum inhibitor benzyloxycarbonyl-Val-Ala-DL-Asp-fluoromethylketone (z-VAD-fmk) or expression of the p35 baculovirus inhibitory protein or overexpression of Bcl-2 inhibits the differentiation process. In addition, z-VAD-fmk amplifies the differentiation-associated production of radical oxygen species in both phorbol ester–differentiated U937 cells and M-CSF–treated monocytes, shifting the differentiation process to nonapoptotic cell death. Altogether, these results indicate that caspase activation specifically contributes to the differentiation of monocytes into macrophages, in the absence of cell death.
Stem cell factor induces eosinophil activation and degranulation: mediator release and gene array analysis
Authors: Oliveira Sandra H. P. , Taub Dennis D. , Nagel James , Smith Robert , Hogaboam Cory M. , Berlin Aaron , Lukacs Nicholas W.
Published: November 2002

Eosinophils are effector cells that play an important role in the damage induced by the allergic process by releasing inflammatory mediators and proteolytic factors after activation. Stem cell factor (SCF) is a primary cytokine involved in hematopoiesis and mast cell differentiation, proliferation, and activation. Studies have also indicated that SCF is directly involved in pathogenesis of allergic airway inflammation. In the present study, we examined the ability of SCF to activate murine eosinophils for increased mediator release and up-regulation of chemokines. Initial data demonstrated that eosinophils have significant levels of surface c-kit protein, SCF receptor. SCF-activated eosinophils degranulate and release eosinophil peroxidase and leukotriene C4 in a dose-dependent manner. In addition, SCF was further shown to induce the release of CC chemokines, RANTES, macrophagederived chemokine (MDC), macrophage inflammatory protein-1β (MIP-1β), and C10 from eosinophils. To identify the extent of SCF-induced activation of eosinophils, we also performed gene array analysis using an array containing 1153 genes related to inflammation, including cytokines and their receptors, growth factors, structural and cytoskeletal genes, signal transduction genes as well as several other classes related to immune/inflammatory responses. The gene analysis indicated that more than 150 genes were significantly up-regulated in eosinophils after SCF stimulation. The gene array results were verified using a quantitative real-time polymerase chain reaction analysis to identify the expression of several chemokine and chemokine receptor genes. Altogether, these studies indicate that SCF is a potent eosinophil degranulator and activator that may play a number of roles during an inflammatory/immune response.
Cdc42/Rac1-dependent activation of the p21-activated kinase (PAK) regulates human platelet lamellipodia spreading: implication of the cortical-actin binding protein cortactin
Authors: Vidal Catherine , Geny Blandine , Melle Josiane , Jandrot-Perrus Martine , Fontenay-Roupie Michaëla
Published: November 2002

Platelet activation by thrombin or thrombin receptor-activating peptide (TRAP) results in extensive actin reorganization that leads to filopodia emission and lamellae spreading concomitantly with activation of the Rho family small G proteins, Cdc42 and Rac1. Evidence has been provided that direct binding of Cdc42-guanosine triphosphate (GTP) and Rac1-GTP to the N-terminal regulatory domain of the p21-activated kinase (PAK) stimulates PAK activation and actin reorganization. In the present study, we have investigated the relationship between shape change and PAK activation. We show that thrombin, TRAP, or monoclonal antibody (MoAb) anti-FcγRIIA IV.3 induces an activation of Cdc42 and Rac1. The GpVI ligand, convulxin (CVX), that forces platelets to lamellae spreading efficiently activates Rac1. Thrombin, TRAP, MoAb IV.3, and CVX stimulate autophosphorylation and kinase activity of PAK. Inhibition of Cdc42 and Rac1 with clostridial toxin B inhibits PAK activation and lamellae spreading. The cortical-actin binding protein, p80/85 cortactin, is constitutively associated with PAK in resting platelets and dissociates from PAK after thrombin stimulation. Inhibition of PAK autophosphorylation by toxin B prevents the dissociation of cortactin. These results suggest that Cdc42/Rac1-dependent activation of PAK may trigger early platelet shape change, at least in part through the regulation of cortactin binding to PAK.
Altered mRNA expression of Pax5 and Blimp-1 in B cells in multiple myeloma
Authors: Borson Nancy D. , Lacy Martha Q. , Wettstein Peter J.
Published: November 2002

Multiple myeloma (MM) is a plasma cell disorder that potentially initiates during an early stage of B-cell development. We encountered an unidentified isoform of B cell–specific activator protein (BSAP, or Pax5) in MM cells while performing differential analyses to compare mRNA expression in malignant and normal plasma cells. Pax5 is a transcription factor that plays a central role throughout B-cell development until the point of terminal differentiation. Our finding of this unique isoform prompted us to investigate Pax5 isoform usage in plasma cells and B-cell populations in other MM and healthy subjects. In contrast to normal Pax5 expression, we observed multiple isoforms of Pax5 in conjunction with low levels of expression of the full-length Pax5 in B cells from MM patients. The expressed isoforms in MM varied considerably from patient to patient, with no clear pattern. We also performed semiquantitative analyses of the mRNA expression levels of B lymphocyte–induced maturation protein (Blimp-1), because expression levels of Pax5 and Blimp-1 have been shown to be inversely correlated. We observed the expression of Blimp-1 in the B-cell populations in all 11 MM patients but in none of 11 healthy subjects. We hypothesize that premature Blimp-1 expression coupled to altered and deficient Pax5 expression causes some proliferating B cells to prematurely differentiate to plasma cells in MM.
Phase II Study of Oxaliplatin, Fluorouracil, and Folinic Acid in Locally Advanced or Metastatic Gastric Cancer Patients
Authors: Louvet C. , André T. , Tigaud J.M. , Gamelin E. , Douillard J.Y. , Brunet R. , François E. , Jacob J.H. , Levoir D. , Taamma A. , Rougier P. , Cvitkovic E. , de Gramont A.
Published: November 2002

PURPOSE: To evaluate the efficacy and safety of an oxaliplatin, fluorouracil (5-FU), and folinic acid (FA) combination in patients with metastatic or advanced gastric cancer (M/AGC).

PATIENTS AND METHODS: Of the 54 eligible patients with measurable or assessable M/AGC, 53 received oxaliplatin 100 mg/m2 and FA 400 mg/m2 (2-hour intravenous infusion) followed by 5-FU bolus 400 mg/m2 (10-minute infusion) and then 5-FU 3,000 mg/m2 (46-hour continuous infusion) every 14 days.

RESULTS: Patients (69% male, 31% female) had a median age of 61 years (range, 31 to 75 years), 89% had a performance status of 0 or 1, 70% had newly diagnosed disease, and 87% had metastatic disease. All had histologically confirmed adenocarcinoma. With a median of three involved organs, disease sites included the lymph nodes (67%), stomach (65%), and liver (61%). A median of 10 cycles per patient and 468 complete cycles were administered. Best responses in the 49 assessable patients were two complete responses and 20 partial responses, giving an overall best response rate of 44.9%. Eight patients underwent complementary treatment with curative intent (six with surgery and two with chemoradiotherapy). Median follow-up, time to progression, and overall survival were 18.6 months, 6.2 months, and 8.6 months, respectively. Grade 3/4 neutropenia, leukopenia, thrombocytopenia, and anemia occurred in 38%, 19%, 4%, and 11% of patients, respectively, and febrile neutropenia occurred in six patients (one episode each). Grade 3 peripheral neuropathy occurred in 21% of patients (oxaliplatin-specific scale). Seven patients withdrew because of treatment-related toxicity.

CONCLUSION: This oxaliplatin/5-FU/FA regimen shows good efficacy and an acceptable safety profile in M/AGC patients, and may prove to be a suitable alternative regimen in this indication.