bluuebunny/crossref_metadata_2025 huggingface dataset:
Pollution induced community tolerance (PICT) and analysis of 16S rRNA genes to evaluate the long‐term effects of herbicides on methanotrophic communities in soil
Authors: Seghers D. , Bulcke R. , Reheul D. , Siciliano S. D. , Top E. M. , Verstraete W.
Published: November 2003

There is an increasing interest in agricultural systems in which the use of herbicides is forbidden. Therefore, soils treated with herbicides atrazine and metolachlor for the last 20 years were compared with soil samples from the same field that had never been treated (control soil). We determined the pollution induced community tolerance (PICT) by evaluating the methane oxidation capacity of soil samples after adding increasing amounts of a methane oxidation inhibitor, 2,4‐dichlorophenoxyacetic acid (2,4‐D). Denaturing gradient gel electrophoresis (DGGE) of 16S rRNA genes assessed whether the soil methanotrophic community differed between the two treatments. Addition of 60 µg 2,4‐D per g soil clearly inhibited methane oxidation in both soils but increased the time needed to oxidize 5% methane in the headspace by 250% for the control soil compared with 175% for the herbicide‐treated soil. This indicates that the soil with a long‐term herbicide history had a greater tolerance to the methane oxidation inhibitor than did the control soil. The DGGE of 16S rRNA genes amplified directly from soil community DNA could also distinguish the two treatments. The banding patterns of the Type I methanotrophs contained fewer bands in the herbicide‐treated soil. It seems that both the PICT approach and DGGE analysis are effective assays to distinguish a long‐term herbicide‐treated soil from an untreated soil.
‘I click, therefore I am (not)’: is cognition ‘distributed’ or is it ‘contained’ in borderless e‐learning programmes?
Authors: Henning Elizabeth
Published: November 2003

This paper will problematise the notion of distributed cognition in courses across cultural learning borders, by presenting a narrative case study of six learners’ journeys to online learning. I will argue that although distributed cognition is a widely acceptable theory in e‐learning, it is based on an assumption that the ‘distribution’ of cognition is reciprocal between toolmakers, texts and users, when perhaps, in certain instances, it excludes the life (and learning) world of local learners in the global, borderless environment. In these instances the cognition that is assumed to be distributed is, in contrast, ‘contained’ in the narrative‐in‐action that is familiar to the learners, whose situated knowledge is embedded and embodied in a non‐global discourse (Henning et al., 2000).
The therapy of gastrointestinal infections associated with the acquired immunodeficiency syndrome
Authors: Wilcox C. M. , Mönkemüller K. E.
Published: November 2003

Although there have been dramatic strides in the therapy of human immunodeficiency virus infection over the last few years, the number of infected people world‐wide is tremendous and, at least in developing countries, continues to expand. Complications which involve the gastrointestinal tract are common in these patients, because the gut is a major site for involvement by opportunistic infections and neoplasms in patients with the acquired immunodeficiency syndrome. It is important to recognize the clinical spectrum of gastrointestinal diseases, as well as the appropriate and most cost‐effective diagnostic strategies, as therapies for a number of these disorders are both widely available and highly effective. This review summarizes the major gastrointestinal infections which are seen in patients with the acquired immunodeficiency syndrome, and their treatment.
Suppressive effect of tetraprenylacetone on gastric atrophy induced by short‐term administration of N‐methyl‐N′‐nitro‐ N‐nitrosoguanidine in rats
Authors: Miwa H. , Nagahara A. , Wada R. , Hirai S. , Endo K. , Watanabe S. , Sato N.
Published: November 2003

Background:

Several studies have been reported on the effects of various therapeutic agents in enhancing or suppressing the carcinogenic activity of N‐methyl‐N′‐nitro‐N‐nitrosoguanidine (MNNG). However, it is still unknown whether a mucosal protective agent could suppress its carcinogenic activity.

Methods:

Twenty‐five Wistar male rats were divided into four groups: group 1, MNNG alone; group 2, MNNG + tetraprenylacetone; group 3, control; group 4, tetraprenylacetone alone. MNNG 100 mg/mL was freely given to groups 1 and 2, and tetraprenylacetone (200 mg/kg intraperitoneal) was additionally administered every other day to the rats in groups 2 and 4. The animals were sacrificed at 10 weeks and the gastric mucosa examined.

Results:

Atrophic changes were observed in the antrum after 8 weeks of oral administration of MNNG. Furthermore, using immunohistological analysis with 5‐bromo‐2′‐deoxyuridine (BrdU), the proliferative zone was found to be enlarged and shifted upward, although the BrdU labelling index of the proliferative zone was unaltered. Intraperitoneal administration of tetraprenylacetone every other day suppressed the MNNG‐induced atrophic change and the alterations proliferative markers. Tetraprenylacetone alone did not have an effect either on morphological or proliferative markers.

Conclusion:

These observations suggest that gastric mucosal defensive factors may play critical roles in suppressing atrophic change inducing carcinogenesis by an exogenic carcinogen.
Furazolidone, amoxycillin, bismuth triple therapy for Helicobacter pylori infection
Authors: Segura A. M. , Gutiérrez O. , Otero W. , Angel A. , Genta R. M. , Graham D. Y.
Published: November 2003

Background:

Metronidazole‐resistant Helicobacter pylori are generally the rule in developing countries such as Colombia. Developing countries need an effective, simple and inexpensive non‐metronidazole therapy for H. pylori infection.

Aim:

To evaluate the combination of bismuth, furazolidone and amoxycillin for the treatment of H. pylori infection in Colombia.

Methods:

Thirty patients with histologically documented H. pylori infection received the combination of bismuth subcitrate 240 mg b.d., furazolidone 100 mg q.d.s. and amoxycillin 500 mg q.d.s. for 14 days. Four or more weeks after ending therapy patients were re‐endoscoped and gastric biopsies were obtained and examined using the Genta stain. Each slide was scored for presence, absence and density of H. pylori, active and chronic inflammation, intestinal metaplasia, erosions and atrophy. Cure was defined as the absence of H. pylori.

Results:

All patients completed the course of therapy. Twenty‐five patients were cured (86%, 95% CI: 65–94%). Mild, well‐tolerated side‐effects were reported by six patients (20%).

Conclusions:

This combination of bismuth, furazolidone and amoxycillin fulfills the criteria for successful H. pylori therapy and appears particularly well suited for developing countries since it is simple, inexpensive and effective. Furazolidone‐containing therapies may become especially useful in the face of a world‐wide increase in H. pylori resistance to metronidazole and macrolides.
NSAIDs upregulate β2‐integrin expression on human neutrophils through a calcium‐dependent pathway
Authors: Fiorucci S. , Santucci L. , Gerli R. , Brunori P. M. , Federici B. , Ugolini B. , Fabbri C. , Morelli A.
Published: November 2003

Background:

Margination of circulating neutrophils (PMN) into the gastric microcirculation is an early and critical event in the pathogenesis of non‐steroidal antinflammatory drug (NSAID)‐induced gastropathy. This effect is mediated through the upregulation of β2 integrins on the PMN surface.

Aims:

To investigate whether indomethacin modulates: (1) Mac‐1 expression; (2) Ca2+ mobilization ([Ca2+]i), protein kinase C and nitric oxide accumulation; and (3) mitogen‐associated protein kinase phosphorylation in human PMN.

Methods:

Human PMN were isolated by centrifugation through a double Ficoll gradient. [Ca2+]i was measured in PMN loaded with fura‐2 and Mac‐1 expression by flow cytometry.

Results:

Indomethacin caused a concentration‐ and time‐dependent upregulation of CD11b and CD18 expression and PMN adhesion to endothelial cells. Maximal upregulation of Mac‐1 expression (40–50%) occurred after a 30‐min incubation with 0.1 mM indomethacin. The effect was prevented by removing the Ca2+. Ionomycin and thapsigargin caused a 7–10‐fold increase in [Ca2+]i and a 2–4‐fold increase in Mac‐1 expression. Indomethacin induced a concentration‐dependent phosphorylation of a 41‐kDa mitogen‐associated protein kinase. Tyrosine kinase inhibitors prevented the effect of indomethacin on Mac‐1 expression and Ca2+ mobilization. Indomethacin and ionomycin increased superoxide generation, myeloperoxidase secretion and PMN adherence to endothelial cells and stimulated nitric oxide production. Indomethacin‐induced Mac‐1 upregulation was prevented by a nitric oxide synthase inhibitor.

Conclusions:

Indomethacin‐induced upregulation of Mac‐1 is mediated by changes in [Ca2+]i and nitric oxide. Phosphorylation of the 41‐kDa mitogen‐associated protein isoform is a previously unreported target of NSAID action. These effects might help to explain the ability of indomethacin to cause gastric neutrophil margination.
Haemodynamic effects of octreotide in portal hypertensive rats receiving propranolol
Authors: MOREAU R. , CAILMAIL S. , GADANO A. , VALLA D. , LEBREC D.
Published: November 2003

Background: The aim of this study was to investigate short‐term effects of propranolol (a non‐selective β‐adrenergic antagonist), octreotide (a long‐acting somatostatin analogue), or a combination of these substances on splanchnic and systemic haemodynamics and arterial blood gases in rats with portal vein stenosis.

Methods: Splanchnic and systemic haemodynamics were measured using the radioactive microspheres method. Eight rats first received an i.v. infusion of isotonic saline (10 μL/min for 15 min) and then an i.v. infusion of octreotide (8 μg.h/kg for 15 min). Eight other rats first received a bolus i.v. injection of propranolol (2 mg) and an i.v. infusion of octreotide 15 min later.

Results: Propranolol or octreotide alone significantly decreased portal pressure (both by 23%), portal tributary blood flow (35 and 10%, respectively) and cardiac index (36 and 26%, respectively). Octreotide administration in rats pretreated with propranolol significantly decreased cardiac index but did not change portal and arterial pressures or portal tributary blood flow. Propranolol significantly increased arterial oxygen tension. Octreotide alone or combined with propranolol significantly decreased oxyhaemoglobin saturation and pH and increased carbon dioxide tension.

Conclusions: In rats with portal vein stenosis, the somatostatin analogue, octreotide, accentuates the short‐term decrease in cardiac index due to propranolol. In addition, octreotide altered arterial blood gases and acid‐base status. In contrast, octreotide does not further decrease portal pressure in animals receiving propranolol.
the screening, diagnosis and optimal management of haemochromatosis
Authors: GEORGE D. K. , POWELL L. W.
Published: November 2003

Haemochromatosis was first recognized as a disease entity over a century ago and its hereditary nature recognized over 60 years ago. However it was only in late 1996 that the haemochromatosis gene was cloned and a single C282Y mutation confirmed as being the cause of all HLA‐linked iron overload in Caucasian populations. Haemochromatosis is common, occurring in approximately 1 in 300 people in Caucasian populations, and untreated can cause serious morbidity and early death. However, the disease remains much underdiagnosed for reasons such as lack of awareness of the disease, the presence of normal liver function tests and the lack or non‐specific nature of symptoms. A␣commercially available DNA‐based test for the haemochromatosis gene is likely to be available in the near future but its place in the diagnosis and management of the disorder is not yet clear. Assessment of body iron stores by measurement of serum ferritin and transferrin saturation, hepatic iron stores and hepatic architecture by liver biopsy will remain important in the future. The haemochromatosis mutation itself has as yet no known influence on morbidity other than via iron loading and organ failure, in particular, hepatic cirrhosis. Thus, diagnosing patients before the development of hepatic cirrhosis is crucial because iron depletion by venesection treatment before the development of cirrhosis results in a normal life expectancy.
Long‐term efficacy of bismuth carbomer enemas in patients with treatment‐resistant chronic pouchitis
Authors: GIONCHETTI P. , RIZZELLO F. , VENTURI A. , FERRETTI M. , BRIGNOLA C. , PERUZZO S. , BELLOLI C. , POGGIOLI G. , MIGLIOLI M. , CAMPIERI M.
Published: November 2003

Background: Mucosal inflammation of the ileal pouch (pouchitis) is the major long‐term complication after ileal pouch‐anal anastomosis for ulcerative colitis. Broad‐spectrum antibiotics are the mainstay of treatment, however, 15% of patients with pouchitis have a chronic, treatment‐resistant disease.

Aim: To determine the safety and efficacy of bismuth carbomer enemas in achieving and maintaining remission in treatment‐resistant chronic pouchitis.

Methods: Twelve patients with treatment‐resistant chronic pouchitis were treated nightly for 45 days with enemas containing elemental bismuth complexed with carbomer. Diagnosis of pouchitis and response to treatment were evaluated with the Pouchitis Disease Activity Index (PDAI), which includes clinical, sigmoidoscopic and histological criteria. Serum bismuth concentrations were determined by atomic absorption.

Results: Ten of 12 patients (83%) went into remission, with a significant decrease of mean total PDAI score from 12 (range 9–15) to 6 (4–15) (P < 0.002), and were continued on bismuth carbomer enemas administered every third night for 12 months. Patients were monitored clinically, sigmoidoscopically and histologically every 2 months for evidence of recurrence (increase ≥ 2 in the clinical symptom portion of the PDAI). Six of 10 patients (60%) were able to maintain remission throughout the 12‐month trial; 4/10 had an exacerbation, two of which occurred soon after discontinuing daily treatment. Serum bismuth levels were negligible in all patients and no side‐effects were registered.

Conclusions: Our findings suggest that bismuth carbomer enemas are safe and effective in achieving and maintaining remission in patients with treatment‐resistant chronic pouchitis.
Gastric corpus IL‐8 concentration and neutrophil infiltration in duodenal ulcer patients
Authors: UEMURA N. , OOMOTO Y. , MUKAI T. , OKAMOTO S. , YAMAGUCHI S. , MASHIBA H. , TANIYAMA K. , SASAKI N. , SUMII K. , HARUMA K. , KAJIYAMA D
Published: November 2003

Background: The purpose of the present study was to examine the association between interleukin‐8 (IL‐8) in the gastric body due to Helicobacter pylori infection and histological gastritis, as well as elucidating the effect of acid secretion inhibitors on H. pylori associated body gastritis in duodenal ulcer patients.

Methods: Twenty H. pylori‐negative patients, 20 H. pylori‐positive patients with chronic gastritis without peptic ulceration, and 20 H. pylori‐positive duodenal ulcer patients (DU) were studied. Four biopsy samples were taken, each from the greater curvature of the antrum and body of the stomach. Biopsies were histologically investigated by ELISA to determine the density of H. pylori, the degree of neutrophil infiltration and the IL‐8 concentration in the mucosa.

Results: In the gastric mucosa of H. pylori‐negative subjects, no IL‐8 and hardly any neutrophil infiltration were observed. In contrast, enhanced IL‐8 production and increased neutrophil infiltration were present in those infected with H. pylori. In H. pylori‐positive patients, a significant correlation was observed between the IL‐8 concentration and the degree of neutrophil infiltration, but no correlation was found in the body mucosa of those with DU. Twelve of 20 DU patients demonstrated hardly any neutrophil infiltration, despite the increased mucosal IL‐8 content in the body. The administration of omeprazole in DU patients markedly increased mucosal neutrophil infiltration even though it did not cause any significant change in the H. pylori density and IL‐8 concentration in the body. Although the effect of omeprazole was transient, a significant increase in neutrophil infiltration continued in comparison with the status before omeprazole administration in those subsequently undergoing maintenance treatment with H2‐blockers.

Conclusion: In H. pylori‐positive chronic gastritis, IL‐8 concentration is enhanced in the mucosa of the body, and is associated with increased neutrophil infiltration. However, in DU patients, despite increases in body IL‐8 concentration, neutrophil infiltration is reduced and the gastritis may be localized in the antrum.
The effect of a single rectal dose of cisapride on delayed gastric emptying
Authors: BRUMMER R.‐J. M. , SCHOENMAKERS E. A. J. M. , KEMERINK† G. J. , HEIDENDAL† G. A. K. , SANDERS‡ D. G. M. , STOCKBRÜGGER R. W.
Published: November 2003

Background: Cisapride has an established prokinetic effect in patients with delayed gastric emptying. However, rectal administration of the drug might be preferred in patients with either dysphagia or nausea due to gastroparesis.

Aim: To determine the effect of a single rectal dose of cisapride 60 mg on gastric emptying in patients with delayed gastric emptying.

Methods: Thirty‐two patients (16 males, 16 females) with demonstrated delayed gastric emptying received a single dose of two suppositories containing either cisapride (2 × 30 mg) or placebo, according to a double‐blind randomized crossover design. Three hours after administration of the suppositories, the patients received a radio‐labelled test meal and radio‐opaque markers for measurement of gastric emptying.

Results: The mean t½ after cisapride administration (76 min, 95% CI: 68–95) was significantly shorter (P = 0.005; n = 28, per‐protocol analysis) than after placebo administration (104 min, 81–126). Four hours after ingestion of the meal significantly fewer radio‐opaque markers remained in the stomach after cisapride than after placebo administration (P < 0.05). Mild to moderate adverse events, mainly involving the gastrointestinal tract, were reported in 10 patients (31%) after cisapride treatment and in four patients (13%) after placebo (N.S.; n = 32).

Conclusion: A single suppository dose of cisapride 60 mg significantly accelerates gastric emptying of the solid phase of a meal and of radio‐opaque markers in patients with previously demonstrated delayed gastric emptying.
Fistula response to methotrexate in Crohn's disease: a case series
Authors: Mahadevan U. , Marion J. F. , Present D. H.
Published: November 2003

Background : Controlled trials have demonstrated the efficacy of methotrexate in the induction and maintenance of remission in luminal Crohn's disease; however, its effect on fistulizing disease is unknown.

Aim : To describe the response to methotrexate therapy in a series of patients with fistulizing Crohn's disease.

Methods : A retrospective chart review was conducted of all patients with Crohn's disease receiving methotrexate in one practice. The response of patients with fistulizing and luminal disease was assessed using clinical and laboratory criteria. Fistula response was categorized as either complete or partial closure.

Results : Thirty‐seven courses of methotrexate therapy were given to 33 patients with luminal and/or fistulizing Crohn's disease. In 16 patients with fistulas, four (25%) had complete closure, five (31%) had partial closure and all had failed or were intolerant to 6‐mercaptopurine therapy. Overall, response to methotrexate was seen in 23 of 37 (62%) treatment courses in patients with luminal and/or fistulizing Crohn's disease. Two of the 33 patients (6%) had a significant adverse event.

Conclusions : In this case series, 56% of patients with Crohn's fistulas on methotrexate showed a complete or partial response to therapy. Further studies are needed to confirm the role of methotrexate alone, and in combination with other therapies, for the treatment of fistulizing Crohn's disease.
Multiple gastric involvement by myeloid antigen CD13‐positive non‐secretory plasma cell leukaemia
Authors: Ohsaka Akimichi , Sato Naotake , Imai Yasufumi , Hirai Shinji , Oka Yuji , Kikuchi Masahiro , Takahashi Atsushi
Published: November 2003

Gastrointestinal tract involvement is a rare complication of plasma cell neoplasia. We present a case of non‐secretory type primary plasma cell leukaemia (PCL) with multiple gastric involvement. Dual surface antigen analysis of bone marrow cells revealed that atypical plasma cells coexpressed CD38 and myeloid antigen CD13. Upper gastrointestinal endoscopy disclosed multiple submucosal masses in the body of the stomach. Endoscopic biopsy specimens showed marked infiltration of atypical plasma cells consistent with a diagnosis of gastric involvement by PCL. Since CD13 antigen is identical to aminopeptidase N, a membrane‐bound glycoprotein thought to be involved in the process of tumour invasion, CD13 expression on neoplastic plasma cells may be related to the gastric involvement in this patient.
Indications for haemopoietic precursor cell transplants in Europe
Authors: Gratwohl A. , Hermans J. , Baldomero H. , Tichelli A. , Goldman J. M. , Gahrton G.
Published: November 2003

Information on 17 206 haemopoietic precursor cell transplants performed in 12 European countries between 1991 and 1993 was used to assess the number of transplants per million inhabitants per year by principal indication and donor source. 16.6 transplants were performed per million inhabitants per year with a range of 9.4–27.7. Differences between countries may be due to the availability of beds and resources or to divergent opinions about treatments. We used the coefficient of variation (CV) to assess the degree of agreement between clinicians with regard to different procedures and indications, with a cut‐off of ≤45% to indicate consensus. The rate of allogeneic transplantation between the 12 countries was more ‘homogenous’ than that of autologous transplantation (mean 6.8 per million inhabitants, range 5.2–9.5, CV 20% versus mean 9.8 per million inhabitants, range 3.6–18.4, CV 40%). Consensus was found for allografting in chronic myeloid leukaemia (CML) in first chronic phase (CV 19%), CML in later phases (CV 31%), acute myeloid leukaemia (AML) in first complete remission (CV 41%), AML beyond first complete remission (CV 34%), acute lymphoblastic leukaemia beyond first complete remission (23%), and severe aplastic anaemia (29%). Consensus for autografting was observed for Hodgkin's disease (CV 44%), non‐Hodgkin's lymphoma (CV 44%), and AML beyond first remission (CV 41%). With this method an assessment of medical opinion can be made in spite of the different availabilities of resources. These data reflect the state of the art in haemopoietic precursor cell usage in Europe; they provide a basis for patient counselling and health‐care planning.
Motility of rhG‐CSF‐induced neutrophils in patients undergoing chemotherapy: evidence for inhibition detected by image analysis
Authors: Azzarà Antonio , Carulli Giovanni , Rizzuti‐Gullaci Angela , Minnucci Sistina , Capochiani Enrico , Ambrogi Fabio
Published: November 2003

The motility of circulating neutrophils from seven patients affected by intermediate and high‐grade non‐Hodgkin's lymphoma was investigated before and after rhG‐CSF administration (5μg/kg/d for 5 d subcutaneously) in the course of chemotherapy. Random motility and bacterial lipopolysaccharide‐induced chemotaxis were studied by the micropore filter technique in a Boyden chamber. These functions were evaluated by a very sensitive technique, based on a computer‐assisted image processing system, capable of giving several parameters about the kinetics of cell migration. Along with a significant increase in neutrophil number, a significant decrease both in random and stimulated motility was found. The kinetics of cell migration showed that the cells maintained the typical gaussian pattern of random motility. On the contrary, neutrophils were found to have lost the typical stimulated migration peak. These findings are consistent with a rhG‐CSF‐induced impairment of the directional movement, rather than of the ability of moving at random. These effects were found in patients who, in the same experimental conditions, had displayed an enhanced phagocytosis and phagocytosis‐associated chemiluminescence along with an enhanced CD32 expression, not due to an aspecific cell manipulation. Two hypotheses may be taken into account: (i) an increased adhesiveness due to a direct or an indirect activity of the cytokine; (ii) an abnormality in the cytoskeleton maturation and/or rearrangement during the accelerated bone marrow transit of myeloid cells. These findings emphasize that rh‐GCSF administration can modulate several functions which play an important role in host defence, and suggest the utility of carrying out further studies to investigate the optimum dosage both to correct neutrophil number and preserve neutrophil functional activities.
Efficacy of recombinant interferon‐alpha (rIFN‐α) in polycythaemia vera: a study of 17 patients and an analysis of published data
Authors: Taylor P. C. , Dolan G. , Ng J‐P. , Paul B. , Collin R. , Reilly J. T.
Published: November 2003

The efficacy and tolerability of rIFN‐α has been evaluated in 17 selected patients with symptomatic polycythaemia vera, diagnosed according to the PRV Study Group criteria. Complete disease control (CR) was achieved, after 1–12 months, in nine patients, with partial control in a further five cases. Three patients failed to respond. Pruritus significantly improved in 83% (10/12) of cases, following 1–28 weeks of treatment. Six patients (35%), however, were unable to tolerate rIFN‐α, on account of weight loss, myalgia and mental changes. Overall, α‐interferon therapy significantly improved venesection requirements, MCV and PCV values, platelet counts, pruritus scores and the degree of splenomegaly. Analysis of pooled published data (100 evaluable patients, including the present study) revealed an overall CR of 60%, a PR of 27%, and a failure rate of 13%. Significant pruritus control (>50% improvement) occurred in 77% of cases. rIFN‐α appears to be an effective therapy for PV‐associated myeloproliferation and/or pruritus, although side‐effects remain a concern. Long‐term studies are now indicated to determine if the natural history of the disease is altered, in particular whether the incidence of myelofibrosis and/or leukaemic transformation is reduced.
Prolonged E‐selectin induction by monocytes potentiates the adhesion of flowing neutrophils to cultured endothelial cells
Authors: Rainger G. E. , Wautier Marie‐Paule , Nash Gerard , Wautier Jean‐Luc
Published: November 2003

We investigated the hypothesis that the infiltration of monocytes into inflamed tissue or damaged vessels would induce a secondary accumulation of neutrophils. Confluent human umbilical vein endothelial cells (HUVEC) and blood monocytes (0.5 or 0.05 monocytes/endothelial cell) were co‐incubated for 4 or 24 h. The adhesion of neutrophils flowing over HUVEC was then analysed by video microscopy. Co‐incubation caused up to a 40‐fold increase in neutrophil adhesion, dependent upon monocyte/HUVEC ratio and duration of incubation. At the lower monocyte/HUVEC ratio, rolling adhesion alone was induced after 4 h co‐incubation; however, the full repertoire of rolling, immobilization and migration of neutrophils was observed at all other combinations of co‐culture ratio and exposure time. After maximal stimulation by monocytes, antibody blockade of the neutrophil integrin CD18 inhibited neutrophil arrest and migration and revealed underlying rolling adhesion. Rolling was supported by endothelial E‐selectin as demonstrated by the almost total abolition of adhesion by a blocking antibody. In a direct comparison, monocytes, tumour necrosis factor α (TNF‐α) and interleukin‐1β (IL‐1β) were assessed for their ability to induce endothelial expression of E‐selectin. E selectin was significantly increased by all agents at 4 h, but monocytes alone were able to maintain high levels of E‐selectin expression for 24 h. We conclude that monocytes can induce prolonged neutrophil adhesion and migration by activating endothelial cells and causing expression of E‐selectin.
Studies of P‐glycoprotein in chronic myelogenous leukaemia patients: expression, activity and correlations with CD34 antigen
Authors: Turkina A. G. , Baryshnikov A. Yu , Sedyakhina N. P. , Folomeshkina S. V. , Sokolova M. A. , Choroshco N. D. , Stavrovskaya A. A.
Published: November 2003

Over‐expression of the P‐glycoprotein (Pgp), transmembrane drug efflux pump, has been shown to cause multidrug resistance of tumour cells (MDR). To investigate the clinical significance of Pgp expression for chronic myeloid leukaemia (CML) diagnosis and monitoring we have studied 38 CML patients in various phases of the disease (chronic phase, CP; accelerated phase, AP; blast crisis, BC). Anti‐Pgp monoclonal antibody UIC2 and FACScan analysis were used. Pgp functional activity was investigated by evaluation of verapamil influence upon rhodamine 123 efflux from the cells. Correlations between Pgp and CD34 expression were investigated. In CP, Pgp‐expressing cells were found in 2/14 patients; in one of them Pgp proved to be non‐functional. There were few Pgp‐expressing cells in AP cases. The group of BC patients consisted of cases resistant to chemotherapy. This gave us the opportunity to consider whether drug resistance of BC CML patients is preferentially connected with Pgp‐mediated MDR. 11/22 BC patients had 20% or more of Pgp‐expressing blasts in the peripheral blood. In all four Pgp+ BC cases studied for Pgp activity this protein was functional. Only 4/22 BC patients demonstrated large (40% or more) fractions of Pgp+ blasts. Moreover, sequential studies of 11 BC CML patients during treatment revealed an increase in the number of Pgp‐expressing cells in only two cases. This suggests that Pgp+ cells did not often accumulate in BC CML patients due to chemotherapy and are the cause of drug resistance in only a few cases. A positive correlation between Pgp and CD34 expression was found (r = 0.69; P = 0.0004). 3/22 BC CML patients had large fractions of both Pgp+ and CD34+ blasts in their peripheral blood. The BC CML patients with this immunophenotype of blast cells may represent a subtype of BC CML resistant to treatment due to Pgp over‐expression.
Bleeding disorder due to platelet prostaglandin H synthase‐1 (PGHS‐1) deficiency
Authors: Matijevic‐Aleksic Nevenka , McPhedran Peter , Wu Kenneth K.
Published: November 2003

Defective platelet prostaglandin H synthase (PGHS) activity has been recognized as a cause of bleeding disorders, but the defect has not been characterized. We evaluated three female patients aged 37, 48 and 55 who presented with a mild bleeding disorder due to platelet dysfunction. None of the patients had underlying diseases or reported use of aspirin or other nonsteroidal anti‐inflammatory drugs. Coagulation screening tests and platelet count were normal in each patient. Platelet aggregation in response to adenosine diphosphate (ADP), collagen and epinephrine were subnormal, characterized by an abnormal second‐wave aggregation and propensity for disaggregation. Arachidonate‐induced platelet aggregation was defective, whereas PGH2‐induced aggregation was normal. Platelet thromboxane A2 (TXA2) production in response to arachidonic acid was reduced in all three patients, i.e. 11.7, 4.6 and 4.4 ng TXB2/3  108 plt respectively (normal range was 49–81 ng/3 10 8 plt), whereas they were normal in response to exogenous PGH2, i.e. 71.4, 56.6 and 48.9 ng/3  108 plts, respectively (normal range 49–85 ng/3  108 plt). These results are consistent with a deficiency of platelet PGHS activity. The level of the constitutive platelet PGHS‐1 and TXA2 synthase (TXAS) proteins were determined on platelet microsomal fractions by Western blot analysis using affinity‐purified polyclonal antibodies highly specific for human PGHS‐1 and TXAS, respectively. In two patients the 70 kD PGHS‐1 protein was undetectable, whereas it was normal in the third patient. The 60 kD TXAS band was normal in all three patients. These findings indicate that human platelet PGHS‐1 deficiency is due to two types of enzyme defects: type 1 defect is manifested by an undetectable PGHS‐1 protein in platelets whereas the type 2 defect is manifested by a normal quantity of PGHS‐1 protein which has an impaired catalytic activity.
Determinants of haemoglobin level in steady‐state homozygous sickle cell disease
Authors: Serjeant Graham , Serjeant Beryl , Stephens Adrian , Roper David , Higgs Douglas , Beckford Marjorie , Cook James , Thomas Peter
Published: November 2003

High total haemoglobin levels in homozygous sickle cell (SS) disease are a risk factor for painful crises, avascular necrosis of the femoral head, proliferative sickle retinopathy, and the acute chest syndrome. Since lowering the haemoglobin level may ameliorate these features, understanding the determinants of total haemoglobin may be of practical importance. A range of possible determinants including red cell characteristics, reticulocytes, serum iron, transferrin saturation, serum ferritin, alpha thalassaemia status, red cell mass and plasma volume, oxygen affinity, red cell survival, transferrin receptor and erythropoietin levels have been measured in 62 patients selected to provide a range of total haemoglobin and fetal haemoglobin levels. There were weak negative associations of haemoglobin with mean cell volume and mean cell haemoglobin concentration, strong negative associations with proportional reticulocyte counts, oxygen affinity, plasma volume, serum transferrin receptors, and erythropoietin levels and strong positive associations with red cell mass. Weighted analysis suggested that the statistically independent determinants of haemoglobin level were alpha thalassaemia, sex, red cell mass/body weight, plasma volume/body weight, fetal haemoglobin, and red cell count. The apparent contributions of red cell survival, P50, reticulocyte count, serum transferrin receptor and erythropoietin levels were explained by the effects of these other variables. The independent determinants as a group explained 91% of the variation in haemoglobin level.
Glass slide smears are a suitable source for RT‐PCR‐based analysis of chromosomal aberrations in leukaemias
Authors: Schoch R. , Jenisch S. , Haferlach T. , Müller‐Ruchholtz W. , Gassmann W. , Löffler H.
Published: November 2003

The polymerase chain reaction (PCR) is a well‐established method to detect cytogenetic abnormalities. The handling of fresh specimens is difficult, therefore a method to use smears of blood or bone marrow as a source would be advantageous. Furthermore, such a technique would give the opportunity to investigate retrospectively bone marrow smears in leukaemias without cytogenetic results. The aim of the present study was to investigate the influence of staining procedures and laboratory handling of smears. We chose CML cases as a model. We demonstrated that smears are a suitable source for PCR without loss of information caused by previous routine laboratory handling.
Thrombin‐mediated activation of endogenous factor XI in plasma in the presence of physiological glycosaminoglycans occurs only with high concentrations of thrombin
Authors: Wuillemin Walter A. , Mertens Koen , Ten Cate Hugo , Hack C. Erik
Published: November 2003

The variable bleeding tendency associated with a genetic deficiency of factor XI (FXI) and the lack of bleeding disorders in individuals with a genetic deficiency of factor XII (FXII) suggest an alternative mechanism for FXI activation in vivo. Recently, thrombin has been shown to activate FXI. However, in plasma this activation has been shown to occur only with exogenous FXI and a non‐physiological cofactor (sulphatides), and the occurrence of this reaction in a plasma environment has been questioned.

Using recently developed sensitive assays for FXIa–inhibitor complexes we found thrombin‐mediated and FXII‐independent activation of endogenous FXI in plasma in the presence of heparan sulphate, heparin, dermatan sulphate or dextran sulphate. Using heparan sulphate, which is present in the human vascular system, activation of about 1–2% of plasma FXI was observed, however, only after addition of very high amounts (500 nmol/l) of human α‐thrombin to FXII‐deficient plasma (at a 1 to 4 final dilution).

We conclude that endogenous FXI in plasma can be activated by thrombin in the presence of various glycosaminoglycans, including the physiological compounds heparan sulphate and dermatan sulphate, but only at very high concentrations of thrombin, corresponding to 100% prothrombin activation in undiluted plasma.
Five unknown mutations in the LR pyruvate kinase gene associated with severe hereditary nonspherocytic haemolytic anaemia in France
Authors: Rouger HervÉ , Valentin Colette , Craescu Constantin T. , GalactÉros FrÉdÉric , Cohen‐Solal Michel
Published: November 2003

A survey of PK‐deficient patients by molecular biology techniques has been performed in France in 26 unrelated families, in which at least one mutation has been characterized. The patients, of European or North African origin, exhibited approximatively 10% of PK activity. Among the PK‐R mutants described, mutation G1529 → A (Arg‐509 → Gln) was the most frequent. The strategy followed for the description of PK mutants in France firstly involves determination of this mutation by PCR amplification and restriction enzyme digestion and, secondly, the sequencing of the gene for negative samples. Study of the mutation at residue 509 in 26 unrelated families indicated that 10/52 defective alleles possessed this mutation. Our study described seven different mutations; five of these have not as yet been documented. Two frameshift mutations were found: the deletion of one G base in a repetition of four Gs in position 1231–1234 (PK Mondor), del C‐1527 (PK Rouen), and three missense mutations: G382 → C (Ala‐114 → Pro) (PK Val‐de‐Marne), C398 → T (Ser‐119 → Phe) (PK Beaujon), A1217 → G (Asn‐392 → Ser) (PK Paris). Two mutations which were detected have been reported previously: C760 → T (Glu‐240 → End) and G1529 → A (Arg‐509 → Gln).
Two regulation points for differentiation in the cell cycle of human megakaryocytes
Authors: Yoshino Takeshi , Sakaguchi Mamoru , Masuda Tetsuya , Kawakita Makoto , Takatsuki Kiyoshi
Published: November 2003

To examine the relationship between cell cycle progression and differentiation in megakaryocytes, variances in the cell cycle during PMA (phorbol 12‐myristate‐13‐acetate)‐induced differentiation were analysed in synchronized growing CMK cells. The cells stimulated with PMA in early S phase showed cell cycle arrest in G2 phase. In addition, the cells stimulated with PMA in early G1 phase showed cell cycle arrest in late G1. The expression of gpIIb/IIIa, megakaryocytic differentiation marker, was markedly induced in G2 phase when the cells were stimulated in early S phase, and was also induced in late G1 phase when cells were stimulated in early G1 phase. Therefore the induction of gpIIb/IIIa expression seems to be associated with cell cycle blockage in both G2 and late G1. Expression of the transcription factor GATA‐1 in cells stimulated in early S phase was much higher than that in control cells at G2 phase. Furthermore, GATA‐1 expression in cells stimulated in early G1 tended to be higher than that in controls at late G1. These periods corresponded to the time that the cell cycle arrest and gpIIb/IIIa expression were induced by PMA. These results suggest that the cell cycle in human megakaryocytic lineage cells may have two regulation points for differentiation.
HELLP syndrome associated with factor V R506Q mutation
Authors: Brenner Benjamin , Lanir Naomi , Thaler Israel
Published: November 2003

The pathogenesis of HELLP (haemolysis, elevated liver enzyme and low platelet count) syndrome, a severe presentation of pre‐eclampsia, is still an enigma. Activated protein C resistance resulting from a mutation in coagulation factor V has recently emerged as the leading cause of thrombosis in pregnancy. We report on two patients with HELLP syndrome who were found to be heterozygous for factor V R506Q mutation, leading to activated protein C resistance. These findings suggest that the pathogenesis of HELLP syndrome is associated with a thrombotic process, and point to the potential benefit of anti‐thrombotic therapy in this condition.
Development and clinical application of a new ELISA assay to determine plasmin–α2‐antiplasmin complexes in plasma
Authors: Montes R. , Páramo J. A. , AnglÈs‐Cano E. , Rocha E.
Published: November 2003

Plasmin–α2‐antiplasmin complexes (PAP) are considered good markers of fibrinolytic activation in vivo. The presence of neoantigens in these complexes offers the possibility to develop specific immunoassays to determine PAP levels. We have developed a sensitive PAP purification method in vitro by adding urokinase to fresh plasma followed by affinity chromatography to lysine‐sepharose and elution with ε‐aminocaproic acid. This material, characterized by SDS‐PAGE and Western blotting, was used to raise monoclonal antibodies (MoAbs). We describe a new enzyme‐linked immunosorbent assay (ELISA) to quantify PAP complexes in plasma. The assay follows the sandwich principle and is based on two MoAbs, CPL12 and CPL15, that bind to the modified α2‐antiplasmin moiety and the plasmin moiety of the complex respectively. The calibration curve was constructed with definite concentrations of purified PAP. The lower limit of the assay is 75 ng/ml and the variation coefficients are 3.5% (intra‐assay) and 10.6% (interassay). A mean value of 573.5 ± 131.4 ng/ml was obtained from PAP concentration in a healthy population (n = 30). Significantly higher PAP levels were observed under diverse clinical conditions in which fibrinolysis is activated: clinical sepsis, acute myocardial infarction (AMI), malignancy, diabetes, pregnancy, elderly people and thrombolytic therapy. From our results we conclude that this ELISA is suitable to measure in vivo plasma PAP levels.
Levels of mdr1 and mrp mRNA in leukaemic cell populations from patients with acute myelocytic leukaemia are heterogenous and inversely correlated to cellular daunorubicin accumulation
Authors: Xu D. , Knaust E. , Pisa P. , Palucka K. , Lundeberg J. , Areström I. , Peterson C. , Gruber A.
Published: November 2003

Multidrug resistance gene (mdr1) expression is associated with a poor prognosis in acute myelocytic leukaemia (AML). Whether expression of the recently described multidrug resistance‐associated gene (mrp) has any prognostic importance in AML is still unclear. The aim of the present study was to investigate the functional role of the mdr1 and mrp mRNA levels in peripheral leukaemic cell populations from patients with AML. Peripheral leukaemic cells from 10 patients with AML were incubated with daunorubicin (DNR). Cellular DNR content was analysed with a fluorescence‐activated cell sorter (FACS). From each cell population the 20–25% cells with the lowest and highest DNR content were sorted out, and mdr1 and mrp RNA were quantified in these subpopulations with competitive polymerase chain reaction. The ratio between the mean DNR content in the cell populations with high and low DNR content varied between 1.9 and 6.6. The cell fraction with low DNR content had higher (3.8–40 times) mdr1 mRNA levels in 10/10 patients and higher (1.4–26 times) mrp mRNA levels in 8/10, as compared to the cell fraction with high DNR accumulation.

In conclusion, mdr1 and mrp mRNA expressions are heterogenous in leukaemic cell populations from patients with AML. The mdr1 expression, and to some extent mrp expression, is inversely correlated to DNR accumulation in vitro
Acute myeloid and T‐cell acute lymphoblastic leukaemia with aberrant antigen expression exhibit similar TCRδ gene rearrangements
Authors: Schmidt C. A. , Przybylski G. , Tietze A. , Oettle H. , Siegert W. , Ludwig W. D.
Published: November 2003

TCRδ gene recombination patterns were analysed by Southern blot, polymerase chain reaction and nucleotide sequencing in acute myeloid leukaemias with coexpression of lymphoid antigens (Ly+ AML, n = 10) as well as in early T‐cell acute lymphoblastic leukaemias with (My+ T‐ALL, n = 10) and without coexpression of myeloid antigens (My− T‐ALL, n = 9). These 29 acute leukaemias exhibiting TCRδ gene rearrangements were selected from 66 Ly+ AML, 14 My+ T‐ ALL and 12 My− T‐ALL cases.

Similar recombination patterns, namely Dδ2Jδ1 and Vδ1Jδ1 gene rearrangements, were observed in Ly+ AML and My+ T‐ALL. In contrast to Vδ2Dδ3 rearrangements in B‐cell precursor ALL, these rearrangements require activation of a T‐cell‐specific TCRδ enhancer. Comparison of My+ T‐ALL and Ly+ AML with My− T‐ALL exhibited a higher incidence of incomplete Dδ2Jδ1 rearrangements in My+ T‐ALL and Ly+ AML. Since a Dδ2Jδ1 rearrangement is an early event in TCRδ recombination, these leukaemias seem to be arrested at an earlier stage of differentiation. Similar patterns of TCRδ rearrangements in My+ T‐ALL and Ly+ AML suggest existence of a common myeloid/T‐lymphoid progenitor cell.

Although weak or missing expression of terminal deoxynucleotidyl transferase (TδT) was found in 7/10 Ly+ AML cases, no difference was observed in numbers of N‐nucleotides inserted in junctional regions when comparing with 3/10 cases exhibiting TdT expression. Since TdT activity is necessary for N‐nucleotide addition, this finding suggests down‐regulation of TδT expression after rearrangement took place in these Ly+ AML cases.
Evidence of involvement of cytotoxic antibodies directed against patient’s HLA class II produced by transfused donor‐derived B cells in post‐transfusion graft‐versus‐host disease
Authors: Nishimura Motoko , Uchida Shigeharu , Mitsunaga Shigeki , Tokunaga Katsushi , Tadokoro Kenji , Juji Takeo
Published: November 2003

Post‐transfusion graft‐versus‐host disease (PTGVHD) is one of the most severe side‐effects of blood transfusion. To characterize the effector cells causing this disease, we cloned lymphocytes from a PTGVHD patient’s peripheral blood. T‐cell and B‐cell clones were established, the origins of which were proven to be transfused donor lymphocytes. It was found that the B cells produced IgG that mediated complement‐dependent cytotoxicity to the cells bearing the patient’s HLA class II genotype. Our results suggest, for the first time, the involvement of B‐cell‐produced cytotoxic antibodies directed against patient’s HLA class II in the pathogenesis of PTGVHD.
Karyotype conversion in two patients with chronic myeloid leukaemia after busulphan‐induced marrow hypoplasia
Authors: Xue Yongquan , Zhou Jin , Yu Fei , Gu Jianming , Guo Yu , Xie Xing , Lin Baojue
Published: November 2003

We report two patients with chronic myeloid leukaemia (CML) developing hypoplasia and karyotype conversion after conventional busulphan therapy. Initially, the percentage of Ph‐positive metaphases in marrow for both patients was 100%, which steadily diminished up to a complete disappearance in case 1 and decreased dramatically in case 2 following hypoplasia. Thereafter Southern blot and RT‐PCR assays revealed no abnormalities. Both patients have survived 9 years and remained in good clinical and haematological remission without any treatment until recently.

We believe that the high sensitivity to busulphan therapy resulted in hypoplasia and karyotype conversion, which contributed to prolonged survival.