bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of plastic spacer handling on salbutamol lung deposition in asthmatic children
Authors: Lipworth Brian J. , Lee Daniel K. C. , Anhøj Jacob , Bisgaard Hans
Published: March 2003

Aims  To study the effects of electrostatics in a plastic spacer on the lung deposition of salbutamol in asthmatic children.

Methods  Twenty‐five children (5–12 years) with mild asthma were given salbutamol hydrofluoroalkane pressurized metered dose inhaler 400 µg via a 750 ml plastic spacer on separate days. Blood samples were taken for plasma salbutamol at 5, 10, 15 and 20 min after inhalation to measure lung bioavailability as a surrogate for relative lung dose. With immediate inhalation following actuation, a new rinsed spacer (NewRinsed ) was compared with a used spacer after repeated daily use (Used ), a spacer rinsed after repeated use (UsedRinsed ) and a spacer primed with benzalkonium chloride to avoid electrostatics (Primed1). In addition, spacers were evaluated using a 15 s inhalation delay following actuation with primed (PrimedDelay) and rinsed (RinsedDelay) spacers. Data were log transformed and expressed as geometric mean fold difference for the average plasma salbutamol concentration (Cav) over 20 min.

Results  There were significant differences (P < 0.05) in Cav (as geometric mean fold difference and 95% CI) between Primed1 vs NewRinsed 1.92 fold (95% CI 1.15, 3.20) and between Used vs NewRinsed 1.75 fold (1.11, 2.76). There were no significant differences comparing Primed1, Used or UsedRinsed. There were also significant differences (P < 0.05) between Primed1 vs PrimedDelay 2.34 fold (1.31, 4.19), or vs RinsedDelay 3.59 fold (2.15, 5.99); and for Used vs PrimedDelay 2.14 fold (1.24, 3.69), or vs RinsedDelay 3.28 fold (2.13, 5.04).

Conclusions  The relative lung dose of salbutamol from a plastic spacer may differ considerably depending on spacer handling suggesting that nonelectrostatic spacers may be the best way forward.
Receptor pharmacogenetics: relevance to CNS syndromes
Authors: Shaikh Sanober , Kerwin Robert W.
Published: March 2003

Pharmacogenetic research dedicated to the investigation of inherited factors that influence drug response has produced exciting results over the past decade. Adding to the knowledge that genetic variation in metabolic enzymes may cause drug‐related adverse reactions, recent studies indicate that variation in neurotransmitter receptors can also be the cause of treatment failure. In addition, recent studies have attempted to use genetic information for the prediction of treatment outcome. The aim of this review is to summarize the most significant findings in pharmacogenetic research in relation to CNS drugs and to outline how these studies could lead to the individualization of drug treatment.
Pharmacokinetics and electrocardiographic pharmacodynamics of artemether‐lumefantrine (Riamet®) with concomitant administration of ketoconazole in healthy subjects
Authors: Lefèvre Gilbert , Carpenter Polly , Souppart Claire , Schmidli Heinz , McClean Mark , Stypinski Daria
Published: March 2003

Aims  To evaluate whether the potent CYP3A4 inhibitor ketoconazole has any influence on the pharmacokinetic and electrocardiographic parameters of the antimalarial co‐artemether (artemether‐lumefantrine) in healthy subjects.

Methods  Sixteen subjects were randomized in an open‐label, two period crossover design study. Subjects received a single dose of co‐artemether (day 1) either alone or in combination with multiple oral doses of ketoconazole (400 mg on day 1 followed by 200 mg o.d. for 4 additional days). Serial blood samples were taken and assayed for artemether and its main active metabolite dihydroartemisinin (DHA), and lumefantrine.

Results  The pharmacokinetics of artemether, its metabolite DHA, and lumefantrine were influenced by the presence of ketoconazole. AUC(0,∞) was increased from 320 to 740 ng ml−1 h (ratio 2.4, 90% CI 2.00, 2.86) for artemether, from 331 to 501 ng ml−1 h (ratio 1.7, 90% CI 1.40, 1.98) for DHA, and from 207 to 333 µg ml−1 h (ratio 1.7, 90% CI 1.23, 2.21) for lumefantrine in the presence of ketoconazole. Cmax also increased in similar proportions for the three compounds (ratio 2.2 (90% CI 1.78, 2.83), 1.4 (90% CI 1.12, 1.74), and 1.3 (90% CI 0.96, 1.64), respectively). The terminal elimination half‐life was increased for artemether (2.5 vs 1.9 h, 90% CI 1.12, 1.72) and DHA (3.1 vs 2.1 h, 90% CI 0.02, 3.36), but remained unchanged for lumefantrine (88 vs 95 h, 90% CI 0.81, 1.04). These increases in exposure to the antimalarial combination were much smaller than observed with food intake (up to 16 fold), and were not associated with increased side‐effects or changes in electrocardiographic parameters. The study medications were well tolerated.

Conclusions  The concurrent administration of ketoconazole with co‐artemether led to modest increases in artemether, DHA, and lumefantrine exposure in healthy subjects. Dose adjustment of co‐artemether is probably unnecessary in falciparum malaria patients when administered in association with ketoconazole or other potent CYP3A4 inhibitors.
Failure of prescribers to adjust antibiotic dose to impaired renal function in daily clinical practice
Authors: Vogtländer N. P. J. , Van Kasteren M. E. F. , Natsch S. , Kullberg B. J. , Hekster Y. A. , Van Der Meer J. W. M.
Published: March 2003

Dose adjustment of antibiotics to renal function is recommended for many antibiotics, which are eliminated by the kidney. Avoiding dose adjustment to renal function leads to unnecessary high plasma concentrations, adverse drug reactions, costs and workload for the nurses.

The aims were to evaluate adherence to hospital guidelines concerning dose adjustment, investigate possible barriers to follow up guidelines, and addressing them through a multifaceted intervention aimed at improving adherence.

The study was performed at a university hospital (953 beds). Data were collected in a pre‐intervention and a post‐intervention period of 3 months each. All consecutive patients with a first prescription of antibiotics at the wards of internal medicine, surgery and neurology were included. Renal function was calculated according to Cockroft & Gault's formula [1]. A table for dose adjustment of antibiotics to renal function was available for every doctor in the written antibiotic guidelines of the hospital.

Sixty‐nine of 225 (31%) patients in the pre‐intervention period had an impaired renal function (renal clearance <50 ml min−1). They received 168 antibiotic prescriptions, of which 129 needed dose adjustment. The dose was actually adjusted in 58 of these 129 cases (45%). The risk for non‐adjustment above the age of 65 as well as 75 years was high (odds ratio 3.1 and 3.5, respectively). A serum‐creatinine level below 140 µmol l−1 was also an important factor for non‐adjustment (odds ratio 5.4). Barriers for not adjusting the dose were underestimation of the prevalence of renal insufficiency, and lack of time to calculate actual renal clearance.

An intervention consisting of audit and feedback to prescribers and nurses, peer discussions with opinion leaders, written information to all prescribers, installation of a calculation program on the desktop of all computers on the wards, and distribution of stickers with a table to estimate renal function to paste in the antibiotic formulary was carried out.

In the post‐intervention period, 68 of 224 (30%) patients had impaired renal function. Dose adjustment was necessary in 129 of 171 prescriptions. This was correctly done in 67 (52%) cases.

We discovered a high prevalence of patients with impaired renal function in our hospital (30%). Especially in the elderly with a serum‐creatinine level in the normal range, renal function is overestimated and hence, overdosing of antibiotics takes place. Despite a multifaceted intervention, the rate of dose adjustment to renal function could only slightly been improved from 45% to 52% (not significant). The complexity of current care of these patients, and a high workload for physicians and nurses makes it hard to increase awareness for this problem in daily practice. Electronic support for prescribing linked to laboratory values may be of much help in the future.
Pharmacokinetics of oral betaine in healthy subjects and patients with homocystinuria
Authors: Schwahn Bernd C. , Hafner Dieter , Hohlfeld Thomas , Balkenhol Nina , Laryea Maurice D. , Wendel Udo
Published: March 2003

Aims Large oral doses of betaine have proved effective in lowering plasma homocysteine in severe hyperhomocysteinaemia. The pharmacokinetic characteristics and metabolism of betaine in humans have not been assessed and drug monitoring for betaine therapy is not available. We studied the pharmacokinetics of betaine and its metabolite dimethylglycine (DMG) in healthy subjects and in three patients with homocystinuria.

Methods Twelve male volunteers underwent an open‐label study. After one single administration of 50 mg betaine kg−1 body weight and during continuous intake of twice daily 50 mg kg−1 body weight, serial blood samples and 24 h urines were collected to determine betaine and DMG plasma concentrations and urinary excretion, respectively. Patients were evaluated after one single dose of betaine.

Results We found rapid absorption (t½,abs 00.28 h, s.d. 0.17) and distribution (t½,λ1 00.59 h, s.d. 0.22) of betaine. A Cmax of 0.94 mmol l−1 (s.d. 0.19) was reached after tmax 00.90 h (s.d. 0.33). The elimination half life t½,z was 14.38 h (s.d. 7.17). After repeated dosage, t½,λ1 (01.77 h, s.d. 0.75) and t½,z (41.17 h, s.d. 13.50) increased significantly (95% CI 0.73, 01.64 h and 19.90, 33.70 h, respectively), whereas absorption remained unchanged. DMG concentrations increased significantly after betaine administration and accumulation occurred to the same extent as with betaine. Renal clearance was low and urinary excretion of betaine was equivalent to 4% of the ingested dose. Distribution and elimination kinetics in homocystinuric patients appeared to be accelerated.

Conclusions Betaine plasma concentrations change rapidly after ingestion. Elimination half‐life increased during continuous dosing over 5 days. Betaine is mainly eliminated by metabolism. More pharmacokinetic and pharmacodynamic studies in hyperhomocysteinaemic patients are needed to refine the current treatment with betaine.
MMR vaccine and idiopathic thrombocytopaenic purpura
Authors: Black Corri , Kaye James A. , Jick Hershel
Published: March 2003

Aims  To estimate the relationship between idiopathic thrombocytopaenic purpura (ITP) and the measles, mumps and rubella (MMR) vaccination in children; calculating the relative risk estimate for ITP with in 6 weeks after MMR vaccination and the attributable risk of ITP within 6 weeks after MMR vaccination.

Methods  Using the General Practice Research Database we identified children with a first‐time diagnosis of ITP from a base population of children aged less than 6 years between January 1988 and December 1999. After describing the characteristics of all the children identified with ITP, we focused on cases aged 13–24 months to perform a population‐based, case–control analysis to estimate the relative risk of developing ITP within 6 weeks after MMR vaccination. We also calculated the risk of ITP attributable to the MMR vaccination.

Results  Sixty‐three children with a first time diagnosis of ITP were identified; 23 cases were between 13 and 24 months old. The relative risk estimate for ITP within 6 weeks after MMR vaccination, compared to the combined group of unvaccinated children and children vaccinated with MMR more than 26 weeks previously was 6.3 (95% CI 1.3–30.1). The attributable risk of developing ITP within 6 weeks after MMR vaccination was estimated to be 1 in 25 000 vaccinations (95% confidence interval 21 300, 89 400).

Conclusion  This study confirms the increased risk of ITP within 6 weeks after MMR vaccination. However, the attributable risk of ITP within 6 weeks after MMR vaccination is low.
Allogeneic bone marrow transplantation with T‐cell‐depleted marrow grafts for patients with poor‐risk relapsed low‐grade non‐Hodgkin's lymphoma
Authors: Mandigers  , Raemaekers  , Schattenberg  , Roovers  , Bogman  , Van Der Maazen  , De Pauw  , De Witte
Published: March 2003

We present the clinical results of allogeneic bone marrow transplantation (BMT) with T‐cell‐depleted grafts from HLA‐matched sibling donors in patients with poor‐risk relapsed low‐grade non‐Hodgkin's lymphoma (NHL). Poor risk was defined as relapse within 12 months after or progression during prior treatment. The conditioning regimen consisted of cyclophosphamide and total‐body irradiation with or without additional idarubicin. Donor marrow was depleted of T lymphocytes using counterflow centrifugation. Post‐BMT prophylaxis of graft‐versus‐host disease (GvHD) consisted of cyclosporine A. 15 patients with a median age of 47 years (range 30–57) were transplanted. All patients engrafted. After a median follow‐up of 36 months (range 9–78), 10 patients were alive and in complete remission (CR). Two of them had relapsed after BMT but re‐entered CR following infusions of leucocytes from the original bone marrow donor. Five patients died; causes of death were cardiomyopathy (n = 1), chronic GvHD (n = 1) and infection during chronic GvHD (n = 3). We conclude that allogeneic T‐cell‐depleted bone marrow transplantation is an efficacious treatment for patients with poor‐risk relapsed low‐grade NHL. Infusions of donor leucocytes reinduced CR in the two patients with relapse after BMT.
Haemoglobin J‐Biskra: a new mildly unstable α1 gene variant with a deletion of eight residues (α50–57, α51–58 or α52–59) including the distal histidine
Authors: Wajcman Henri , Dahmane Malika , Préhu Claude , Costes Bruno , Promé Danielle , Arous Nicole , Bardakdjian‐Michau Josiane , Riou Jean , Ayache Kadour Cherif , Godart Christian , Galactéros Frédéric
Published: March 2003

Single point mutation, which accounts for 92% of the 700 known variants, is the most frequent genetic defect responsible for abnormal haemoglobins. Small deletions (or insertions) involving from one to five residues are also observed, but in only approximately 5% of cases. The remaining variants produce fusion or extended haemoglobins. A deletion of eight residues, which included the distal histidine and its neighbours (α50–57, α51–58 or α52–59), was found in Hb J‐Biskra. This new α‐chain variant was mildly unstable in vitro only and there was no haematological or biochemical evidence of haemolysis in the affected family members. 24 nucleotides were missing in a region of the α1 gene showing an identical sequence of eight nucleotides at both ends. Several starting points could therefore lead to the same nucleotide and aminoacid remaining sequence. This deletion is the largest up to now reported in a haemoglobin molecule which is expressed at an almost normal level in the red blood cell. Comparison of the DNA sequences near to the deleted (or inserted) regions in the various haemoglobins carrying this type of abnormality almost always revealed the presence of a sequence that was hypothesized to slow down progression of the replication fork, and of repeats that may lead to possible secondary structures favouring slipped mispairing.
CD8+/CD57+ cells and apoptosis suppress T‐cell functions in multiple myeloma
Authors: Frassanito  , Silvestris  , Cafforio  , Dammacco
Published: March 2003

The aim of this study was to evaluate the role of CD8+/CD57+ lymphocytes in the immune dysregulation of multiple myeloma (MM). Cytofluorimetry of peripheral blood lymphocytes (PBL) purified from 39 MM patients showed an inverse relationship between the percentage of CD8+/CD57+ cells and CD4/CD8 ratio. Analysis of their activation antigens revealed that they were prevalently HLA‐DR+ and Fas+. Removal of CD8+/CD57+ cells from MM PBL significantly improved cell proliferation and pokeweed mitogen (PWM)‐induced polyclonal Ig production in vitro, whereas the addition of supernatants from patients' CD8+/CD57+ cell cultures to normal PBL suppressed both the PWM‐driven Ig synthesis and the proliferative rate of stimulated PBL, supporting the contention that CD8+/CD57+ cells release in vitro an inhibitory factor that is directly involved in T‐cell regulatory function. However, since the proliferative recovery of PWM‐ and phytohaemagglutinin (PHA)‐stimulated MM PBL in the absence of CD8+/CD57+ lymphocytes was only partial, a dysregulated activation‐induced apoptosis was anticipated. In fact, patients' PBL displayed an increased susceptibility to apoptosis and this was significantly enhanced after PWM and, even more, after PHA stimulation. Analysis of CD57 antigen expression on apoptotic or viable cells demonstrated a substantial defect of apoptosis in the CD8+/CD57+ population. Our results indicate that both the immunosuppressive effect of CD8+/CD57+ cells and the enhanced susceptibility to apoptosis of PBL could be involved in the pathogenesis of the immunodeficiency observed in this disease.
Effects of hormone replacement therapies on fibrinogen and plasma viscosity in postmenopausal women
Authors: Fröhlich  , Schunkert  , Hense  , Tropitzsch  , Hendricks  , Döring  , Riegger  , Koenig
Published: March 2003

In women, cardiovascular morbidity and mortality sharply increase after the onset of menopause. There is substantial evidence that hormone replacement therapy (HRT) may decrease the risk of coronary heart disease (CHD); however, the mechanisms of this preventive effect are unclear.

We investigated the association between HRT and plasma viscosity as well as fibrinogen levels in postmenopausal women of a population‐based sample (n=300, age 52–65 years). A total of 94 women used HRT; of these, 50 took oestrogen monotherapy and 44 used oestrogen–progesterone combinations.

HRT was associated with significantly lower fibrinogen concentrations (2.32v2.68 g/l,P<0.001) and decreased plasma viscosity (1.147v1.176 mPa/s,P=0.01). Multivariate analyses controlling simultaneously for the effects of age, smoking, body mass index, and use of diuretics confirmed decreased fibrinogen and plasma viscosity values in women using HRT. A trend towards lower plasma viscosity (1.139v1.160 mPa/s) and plasma fibrinogen (2.28v2.44 g/l) was observed in women on oestrogen–progesterone combinations as compared with oestrogen monotherapy users; however, after controlling for the above‐mentioned variables these differences were not statistically significant.

This study demonstrates decreased plasma viscosity in women on HRT. Improved rheology offers a mechanism by which HRT lowers the risk of CHD in postmenopausal women.
Accumulation of catalytically active PKC‐ζ into the nucleus of HL‐60 cell line plays a key role in the induction of granulocytic differentiation mediated by all‐trans retinoic acid
Authors: Bertolaso Lucia , Gibellini Davide , Secchiero Paola , Previati Maurizio , Falgione Daniela , Visani Giuseppe , Rizzoli Riccardo , Capitani Silvano , Zauli Giorgio
Published: March 2003

The effect of differentiating doses of all‐trans retinoic acid (ATRA, 10−6 m) and vitamin D3 (10−7 m) was investigated on the nuclear levels of endogenous ceramide and protein kinase C‐ζ (PKC‐ζ) catalytic activity in HL‐60 myeloid cells. ATRA induced a parallel increase of ceramide and catalytically active PKC‐ζ into the nuclear compartment of HL‐60 cells (peak at 72 h). On the other hand, vitamin D3 increased the levels of nuclear ceramide and PKC‐ζ activity to a lesser extent and with a delayed kinetics compared to ATRA (peak at 96 h).

Pretreatment of HL‐60 cells with high pharmacological concentrations of exogenously‐added C2‐ceramide (10−6 m) completely blocked the ATRA‐mediated activation of nuclear PKC‐ζ. Exogenous C2‐ceramide (10−6 m) also inhibited the granulocytic differentiation induced by ATRA, whereas it did not affect monocytic differentiation mediated by vitamin D3.

Transient transfection experiments performed with a plasmid construct containing a constitutively active mutated form of the PKC‐ζ cDNA fused in 3′ to a fluorescent tag protein (pEGFP‐PKC‐ζ) demonstrated that the overexpression of catalytically active PKC‐ζ was not accompanied by the appearance of a differentiated morphology. These findings suggest that nuclear PKC‐ζ is necessary but not sufficient to induce granulocytic differentiation of HL‐60 myeloid malignant cells.
Norethisterone therapy for bleeding due to gastrointestinal telangiectases in Glanzmann's thrombasthenia
Authors: Leach  , Makris  , Hampton  , Preston
Published: March 2003

We report a case of a patient with Glanzmann's thrombasthenia and anti‐GPIIb/IIIa alloantibodies who developed life‐threatening and intractable bleeding from gastrointestinal telangiectatic lesions. After a period of transfusion‐dependent gastrointestinal bleeding despite tranexamic acid, oral iron, omeprazole and platelet transfusions, the use of oral norethisterone produced a significant improvement with a marked reduction in her transfusion requirements.
Positive direct antiglobulin tests and haemolytic anaemia following therapy with beta‐lactamase inhibitor containing drugs may be associated with nonimmunologic adsorption of protein onto red blood cells
Authors: Garratty George , Arndt Patricia A.
Published: March 2003

A high incidence (39%) of positive direct antiglobulin tests (DATs) has been reported in patients taking Unasyn [ampicillin sodium plus sulbactam sodium (a beta‐lactamase inhibitor)]. Three of four patients, with positive DATs, receiving Unasyn or Timentin [ticarcillin disodium plus clavulanate potassium (also a beta‐lactamase inhibitor)] developed a haemolytic anaemia (HA) associated with a positive DAT, which resolved when drug therapy was stopped. The patients' sera did not react with red blood cells (RBCs) in the presence of Unasyn or Timentin, but when drug‐treated RBCs were tested, patients' sera and normal sera reacted equally by indirect antiglobulin test. Following incubation in normal sera, RBCs treated with Unasyn, Timentin, Augmentin (amoxicillin + clavulanate), sulbactam and clavulanate reacted with anti‐human globulin and anti‐human albumin (an index of non‐specific adsorption); RBCs treated with ampicillin and amoxicillin were nonreactive.

The beta‐lactamase inhibitors sulbactam and clavulanate seem to cause nonimmunologic adsorption of protein onto RBCs in vitro. This may explain the high incidence of positive DATs detected in patients taking Unasyn, which contains sulbactam. It was not possible to prove that there was a direct association between the nonspecific uptake of protein onto drug‐treated RBCs in vitro with the positive DATs or the HA.
Mutations of the granulocyte‐colony stimulating factor receptor in patients with severe congenital neutropenia are not required for transformation to acute myeloid leukaemia and may be a bystander phenomenon
Authors: Bernard Tanya , Gale Rosemary E. , Evans Jane P. M. , Linch David C.
Published: March 2003

Point mutations of the granulocyte‐colony stimulating factor receptor (G‐CSFR) resulting in an abnormally truncated receptor have been implicated in the pathogenesis of some cases of severe congenital neutropenia (SCN) and in the transformation of SCN to acute myeloid leukaemia (AML). We report here studies in 11 patients with SCN. No mutations were detected in the one patient who developed AML indicating that development of such mutations is not a prerequisite for transformation. Truncation mutations were detected in a minor percentage of transcripts from two other patients. In one patient the mutation has been constant at a low level (5–10% of total mRNA and 2/40 myeloid colonies) for 2 years. In the other patient the mutation was acquired, remained present at low levels for nearly 3 years and then spontaneously disappeared. Both patients had polyclonal haemopoiesis. We hypothesize that these mutations do not cause SCN, are randomly acquired with the mutant clone being expanded to detectable levels by high levels of exogenous or endogenous G‐CSF, and may disappear by clonal succession. In a pre‐leukaemic marrow the mutated subclone could achieve high levels, but this does not necessarily indicate a primary role of the mutant receptor in the leukaemogenic process.
Variable expression of CD3‐zeta and associated protein tyrosine kinases in lymphocytes from patients with myeloid malignancies
Authors: Buggins  , Hirst  , Pagliuca  , Mufti
Published: March 2003

In myeloid malignancies, T‐cell and NK function has been shown to deteriorate with transformation from pre‐leukaemia to advanced disease. Immune dysfunction in solid tumours has been attributed to abnormal signal transduction, possibly through altered expression of intracellular components of the TCR/CD3 complex (e.g. CD3‐ζ), receptors on NK cells and their associated protein tyrosine kinases (PTKs; p56lck, p59fyn and ZAP‐70). Using a flow cytometric method to detect dual‐expression of surface proteins and intracellular components of the TCR/CD3 complex, we have studied 46 patients with myeloid malignancies. CD3‐ζ expression was abnormal in 64% of patients, and was more prominent in those with advanced disease. Three patients with reduced CD3‐ζ were analysed both pre‐ and post‐treatment, and recovery of CD3‐ζ expression was associated with successful remission induction (expression of PTKs was variable and reduced levels were seen all disease stages). The results of this study suggest that loss of signalling proteins is not a result of direct contact of leukaemic cells with lymphocytes per se or the extent of the leukaemia burden, but to a specific property of some myeloid malignancies, which is more frequently acquired with greater malignant transformation.
Prognostic value of cytogenetics in multiple myeloma
Authors: Seong Chumyong , Delasalle Kay , Hayes Kimberly , Weber Donna , Dimopoulos Meletios , Swantkowski Jolynn , Huh Yang , Glassman Armand , Champlin Richard , Alexanian Raymond
Published: March 2003

Karyotypic studies of bone marrow were conducted in 79 previously untreated patients with multiple myeloma who received a standard programme of chemotherapy. An abnormal karyotype was observed in 46% of patients, virtually all showing multiple abnormalities consistent with a long period of preclinical clonal evolution. Patients with an abnormal pattern showed various aberrations with hyperdiploidy in 64%, pseudodiploidy in 5% and hypodiploidy in 31%. The number of chromosomes affected ranged from two to 19 (median 10), with at least one trisomy in 83%, one monosomy in 75%, and one translocation in 42% of patients. Lymphoma‐like karyotypes were present in 17% of patients with an abnormality but were not associated with atypical clinical features, such as an extramedullary mass, leukaemia, or increased serum lactate dehydrogenase. Monosomy or deletion of chromosome 13 was present in 47% of patients with an abnormal pattern, who lived for a shorter duration (median 10 months) than patients with other abnormalities (median 34 months) or with diploidy (median 35 months). The cause of the short survival of patients with monosomy or deletion of chromosome 13 was not clear, but further studies on the relationship with specific oncogenes are indicated.
Factor VII Morioka (FVII L‐26P): a homozygous missense mutation in the signal sequence identified in a patient with factor VII deficiency
Authors: Ozawa Tetsuo , Takikawa Yasuhiro , Niiya Kenji , Ejiri Naoko , Suzuki Kazuyuki , Sato Shunichi , Sakuragawa Nobuo
Published: March 2003

We investigated the molecular basis of factor VII deficiency in a Japanese patient and identified a novel missense mutation in the signal sequence of the gene. Factor VII activity and antigen level measured in the patient were 10.7% and 11% of normal, respectively. All exons except 1B and the 5′‐flanking region containing promoter region were amplified by polymerase chain reaction (PCR) from genomic DNA. Sequencing analysis of the PCR fragments revealed that the patient was a homozygote for a T to C substitution at nucleotide position 38. This mutation predicts an amino acid replacement of leucine to proline at codon −26 in the hydrophobic core of the signal peptide, which probably affects translocation of the protein into endoplasmic reticulum and subsequently causes reduction in plasma factor VII level.
Differentiation‐linked changes in granulocyte‐macrophage colony‐stimulating factor receptor mediated signalling in the HL‐60 promyelocytic cell line
Authors: Wheadon Helen , Roberts Pamela J. , Linch David C.
Published: March 2003

Granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) induces the proliferation and maturation of immature myeloid progenitor cells and primes mature cell function in phagocytes. To investigate whether the biochemical events following the binding of GM‐CSF to its receptor are differentiation dependent we analysed GM‐CSF mediated activation of the JAK 2‐STAT 5 and MAP kinase pathways in undifferentiated HL‐60 cells and HL‐60 cells induced to differentiate with dimethyl sulphoxide (DMSO) or retinoic acid (RA). GM‐CSF stimulated MAP kinase activation in both the undifferentiated and differentiated HL‐60 cells. Activation of MAP kinase (expressed as a proportion of total cellular MAP kinase) was maximal at 5 min and of similar magnitude in both cell types. There was, however, a marked difference in the later kinetics of activation, with the response being transient in the undifferentiated cells and disappearing within 15 min, whereas it was prolonged and persisted for at least 60 min in the differentiated cells. GM‐CSF mediated activation of STAT 5 was markedly increased (15–20‐fold) after differentiation of HL‐60 cells but the kinetics of activation did not change. The increase in STAT 5 activation was not due to a change in total cellular STAT 5 expression but correlated with increased JAK‐2 protein levels. These data show that in the HL‐60 cell model, differentiation modulates the activation of signalling molecules downstream of the GM‐CSF receptor.
Characterization of the thromboxane synthase pathway product 12‐oxoheptadeca‐5(Z)‐8(E)‐10(E)‐trienoic acid as a thromboxane A2 receptor antagonist with minimal intrinsic activity
Authors: Ruf  , Mundkowski  , Siegle  , Hofmann  , Patscheke  , Meese
Published: March 2003

Thromboxane synthase forms thromboxane (TX) A2 and 12(S)‐hydroxyheptadeca‐5(Z)‐8(E)‐10(E)‐trienoic acid (HHT) at equimolar amounts. Twelve‐oxoheptadeca‐5(Z)‐8(E)‐10(E)‐trienoic acid (Oxo‐HT) is the primary metabolite of HHT and has been described to be an inhibitor of platelet aggregation. Functional studies, Schild analysis and competitive binding studies were performed to clarify its mode of action. Oxo‐HT was prepared biosynthetically as well as chemosynthetically, purified and characterized by gas chromatography and mass spectrometry. Platelet activation was assessed by determination of shape change, aggregation, fibrinogen binding and P‐selectin expression using optical aggregometry and flow cytometry. Oxo‐HT 0.1 n
M to 50 μM did not induce platelet activation. Furthermore, it had no effect on platelet activation induced by thrombin, ADP or PAF. In contrast, Oxo‐HT inhibited platelet aggregation, fibrinogen binding and P‐selectin expression induced by U46619 in a competitive manner. Schild analysis for U46619‐induced fibrinogen binding and P‐selectin expression revealed pA2 values of 6.1 and 6.6, respectively, which correspond to Kd values of approximately 0.8 μM and 0.3 μM, respectively. Oxo‐HT also inhibited U46619 induced shape change (IC50 ≃ 10 μM). However, Oxo‐HT over a concentration range of 0.1–1 μM enhanced the partial shape change induced by low concentrations of U46619. Thus Oxo‐HT seems to possess a minimal agonistic potential, which alone is not sufficient to trigger a platelet activation but can enhance low levels of platelet activation. Oxo‐HT blocked the binding of [3H]SQ 29548 in a concentration‐dependent manner, whereas HHT did not displace [3H]SQ 29548. The Kd of Oxo‐HT determined from competition binding studies was 7.7 μM, about 10–25‐fold higher than the apparent Kd determined by Schild analysis. This discrepancy might be due to a desensitization of the TXA2 receptor triggered by the minimal intrinsic activity of Oxo‐HT. We conclude that Oxo‐HT is a naturally occurring specific TXA2 receptor antagonist with minimal intrinsic activity. Oxo‐HT may contribute to the regulation of TXA2‐induced platelet activation in vivo.
Interphase cytogenetics and competitive RT‐PCR for residual disease monitoring in patients with chronic myeloid leukaemia during interferon‐α therapy
Authors: Tchirkov Andrei , Giollant Michel , Tavernier FranÇoise , BrianÇon Georges , Tournilhac Olivier , Kwiatkowski Fabrice , Philippe Pierre , Choufi Bachra , DemÉocq FranÇois , Travade Philippe , Malet Paul
Published: March 2003

There is a need for fast and sensitive methods to evaluate the response of patients with chronic myeloid leukaemia (CML) to interferon‐α (IFN‐α) therapy to complement cytogenetic analysis of Philadelphia (Ph) chromosome‐positive metaphases. We have used interphase FISH (fluorescence in situ hybridization) and competitive RT‐PCR (reverse transcriptase‐polymerase chain reaction) techniques for detection of BCR‐ABL‐positive cells to measure suppression of leukaemic clone in a series of 51 follow‐up samples from 24 CML patients undergoing IFN‐α treatment. Interphase FISH analysis of the malignant clone in bone marrow using BCR and ABL probes was found to be highly correlated to conventional G‐banding metaphase examination (r = 0.98). RT‐PCR quantification of BCR‐ABL mRNA transcripts in blood also showed a high degree of concordance with the proportion of Ph‐positive metaphases (r = 0.93). In addition, the degree of cytogenetic response did not influence the equivalence between karyotype analysis and molecular methods. We concluded that interphase FISH and competitive RT‐PCR provide reliable information on residual tumour burden and response to IFN‐α in CML patients. These molecular methods may significantly improve the efficiency of residual disease monitoring during IFN‐α therapy of CML.
Recombinant human megakaryocyte growth and development factor increases levels of circulating haemopoietic progenitor cells post chemotherapy in patients with acute myeloid leukaemia
Authors: Geissler Klaus , Kabrna Eva , Stengg Susanna , Schwarzinger Ilse , KNÖbl Paul , Valent Peter , Eichinger Sabine , Barge Alan , Lechner Klaus
Published: March 2003

By participating in a randomized safety and efficacy study of pegylated‐recombinant human megakaryocyte growth and development factor (PEG‐rHuMGDF) post induction and consolidation chemotherapy for de novo acute myeloid leukaemia, serial determinations of circulating haemopoietic progenitor cells were performed during 18 chemotherapy courses in eight patients (three receiving placebo; one, 2.5; and four, 5.0 μg/kg/d MGDF, respectively). Whereas failure to achieve complete remission (CR) was generally associated with poor progenitor cell increments following chemotherapy, substantial progenitor cell mobilization consistently occurred during haemopoietic recovery in patients entering, or in CR, with significantly higher peak values in patients receiving 5 μg/kg/d of MGDF as compared to controls. The median increases of progenitor cell numbers by chemotherapy alone and chemotherapy plus 5.0 μg/kg/d MGDF over that in normal individuals with steady‐state haemopoiesis were 10‐ and 45‐fold for CFU‐GM, 3‐ and 17‐fold for BFU‐E, and 2‐ and 18‐fold for CFU‐mix. CFU‐Mk levels were not increased above normal by chemotherapy alone but were 15‐fold enhanced by chemotherapy plus MGDF. Recruitment of CD34+ cells post chemotherapy was also potentiated by MGDF. Our results suggest MGDF as a potent agent to augment progenitor cell mobilization after successful induction or consolidation chemotherapy in patients with AML.
Congenital factor X deficiency: spectrum of bleeding symptoms in 32 Iranian patients
Authors: Peyvandi  , Mannucci  , Lak  , Abdoullahi  , Zeinali  , Sharifian  , Perry
Published: March 2003

The spectrum of the clinical manifestations of congenital factor X deficiency was studied in 32 Iranian patients. The most frequent symptom was epistaxis, which occurred in 72% of patients, with all degrees of deficiency. Other mucosal haemorrhages (e.g. haematuria, gastrointestinal bleeding) were less frequent and occurred mainly in patients with unmeasurable factor X. Menorrhagia occurred in half of the women of reproductive age. Soft tissue bleeding occurred in two‐thirds of the patients; spontaneous haematomas and haemarthroses led to severe arthropathy in five patients. Bleeding from the umbilical stump was an unexpected finding in nine patients. This study demonstrated that the bleeding tendency of factor X deficiency is severe and correlates with factor levels.
In vitro drug‐induced cytotoxicity predicts clinical response to fludarabine in B‐cell chronic lymphocytic leukaemia
Authors: Morabito Fortunato , Stelitano Caterina , Callea Ida , Dattola Antonella , Console Giuseppe , Pucci Giulia , Iacopino Pasquale , Callea Vincenzo , DI Raimondo Francesco , Brugiatelli Maura
Published: March 2003

We investigated the possible value of an in vitro drug‐induced cytotoxicity assay in predicting clinical response to fludarabine (FAMP) in chronic lymphocytic leukaemia (CLL). The median FAMP‐LD50 values for cases designated as complete response (CR), partial response (PR), no response (NR) and progressive disease (PD) were 1.43, 2.05, 24.8 and 77.9 μg/ml, respectively (P=0.013). A significant lower mean FAMP‐LD50 value was observed in the in vivo responding cases as compared to non‐responding ones (3.0 ± 0.82 SEM v 42.3 ± 12.6 SEM, P=0.001). Only FAMP‐LD50  2.5 μg/ml and more than four FAMP courses significantly influenced the response rate in univariate and multivariate analyses. The MTT assay may be of value in predicting clinical response to FAMP in CLL patients.
MDR1 and MRP expression in chronic B‐cell lymphoproliferative disorders
Authors: Webb  , Brun  , Mcniven  , Le Couteur  , Craft
Published: March 2003

The role of the MDR1 and MRP genes in drug resistance in patients with chronic lymphocytic leukaemia (CLL)/non‐Hodgkin's lymphoma (NHL) is unclear. We hypothesized that any relationship between levels of expression and exposure to P‐glycoprotein (P‐gp) transportable drugs may become evident by using a measure of gene expression that combined the number of positive cells and the degree of positivity. 68 CLL/NHL patients were analysed using flow cytometry with MDR1 and MRP specific antibodies and were divided into subgroups, untreated (n = 31), treated with non P‐gp transportable drugs (n = 26), those treated with low total doses of P‐gp transportable drugs (n = 6) and patients treated with high total doses of P‐gp transportable drugs (n = 5). The group exposed to high doses of P‐gp transportable drugs had higher levels of MDR1 expression when compared to all other groups (P < 0.05, ANOVA). A positive correlation between the level of MDR1 expression and the cumulative dose of P‐gp transportable drugs was demonstrated (P = 0.02). MRP expression was higher in those patients exposed to high doses of P‐gp transportable drugs when compared to all other groups (P < 0.05, ANOVA), although only a trend towards a linear dose correlation effect could be established (P = 0.08). We concluded that MDR1 and MRP are involved in drug resistance but only in patients treated with P‐gp transportable drugs.
Granulocyte colony‐stimulating factor‐induced dephosphorylation of a 45 kD cytosolic protein in HL‐60 cells differentiating into neutrophils
Authors: Yamaguchi Teruhide , Oshizawa Tadashi , Yamaguchi Takamasa , Suzuki Kazuhiro , Yamamoto Yukio , Hayakawa Takao
Published: March 2003

Granulocyte colony‐stimulating factor (G‐CSF)‐induced alteration of phosphoprotein during differentiation of HL‐60 cells was studied. From the two‐dimensional gel electrophoresis analysis of phosphoproteins, a 45 kD phosphoprotein in the cytosolic fraction of DMSO‐pretreated HL‐60 cells was rapidly dephosphorylated by the addition of G‐CSF. This 45 kD phosphoprotein migrated into four or five spots between 4.5 and 5.5 pI. The dephosphorylation of 45 kD protein was observed within at least 10 min and reached a maximum at 60 min. Phosphoamino acid analysis showed that only serine residue of 45 kD phosphoprotein was phosphorylated, suggesting that G‐CSF induced an activation of serine phosphatase. Furthermore, Staurosporine and calphostin C inhibited the phosphorylation of 45 kD protein, suggesting that protein kinase C or its downstream kinase(s) is involved in the phosphorylation of 45 kD protein. These results indicate that G‐CSF causes dephosphorylation of a 45 kD cytosolic phosphoprotein which may play a role in signal transduction of G‐CSF.
IDA‐FLAG (idarubicin, fludarabine, cytarabine, G‐CSF), an effective remission‐induction therapy for poor‐prognosis AML of childhood prior to allogeneic or autologous bone marrow transplantation: experiences of a phase II trial
Authors: Fleischhack  , Hasan  , Graf  , Mann  , Bode
Published: March 2003

A phase II trial was designed to explore the potential feasibility and efficacy of a reinduction therapy consisting of fludarabine, cytarabine, idarubicin and granulocyte colony stimulating factor (G‐CSF) for acute myelogenous leukaemia (AML) patients with poor prognosis.

Twenty‐three patients aged 1.2–17.5 years with refractory (n = 3), relapsed (n = 19) or secondary (n = 1) AML were treated with the IDA‐FLAG regimen, a combination therapy of idarubicin (days 2–4, 12 mg/m2/d), fludarabine (days 1–4, 30 mg/m2/d), cytarabine (days 1–4, 2000 mg/m2/d) and G‐CSF (day 0 up to ANC > 1 × 109/l, 400 μg/m2/d). They received a total of 37 courses of IDA‐FLAG and/or FLAG (IDA‐FLAG without idarubicin). 17/23 patients achieved a complete remission (CR) with a median duration of 13.5 months (1–39 months), one patient showed a partial remission, and five were nonresponders while in CR, 11 patients underwent bone marrow or PBSC (peripheral blood stem cells) transplantation. Overall, nine patients remain in continuous complete remission with a median duration of 17.5 months (9.5–39 months). The toxicity of the IDA‐FLAG courses was more severe than for the FLAG courses with marked neutropenia and thrombocytopenia (for IDA‐FLAG: median 22.5 and 25 d respectively; for FLAG: median 10.5 and 14 d respectively). Pulmonary infections were the main nonhaematological toxicity. One patient died in CR from invasive aspergillosis.

The IDA‐FLAG regimen produced a CR of >12 months in more than half of the patients and can be recommended as a therapeutic option prior to allogeneic or autologous bone marrow transplantation.
Targeting of saporin to Hodgkin's lymphoma cells by anti‐CD30 and anti‐CD25 bispecific antibodies
Authors: Sforzini Sabrina , DE Totero Daniela , Gaggero Alessia , Ippoliti Rodolfo , Glennie Martin J. , Canevari Silvana , Stein Harald , Ferrini Silvano
Published: March 2003

CD25 and CD30 represent suitable target molecules for bispecific antibody (bimAb)‐driven toxin delivery to lymphoid tumour cells. We describe two new anti‐CD30/anti‐saporin bimAbs (termed CD30 × sap1 and CD30 × sap2), produced by hybrid hybridomas, which react against non‐cross‐reactive epitopes of the saporin molecule, and compared their effect with a bimAb reacting with saporin and with CD25 (CD25 × sap1). In a protein synthesis inhibition assay these bimAbs were able to enhance saporin toxicity (IC50 8.5 × 10−9M in the absence of mAbs) with a similar activity: in the presence of 10−9 M CD30 × sap1 bimAb the IC50 was 2.75 × 10−11 M, whereas with CD30 × sap2 bimAb the IC50 was 6.5 × 10−11 M and CD25 × sap1 bimAb displayed an IC50 of 3 × 10−11 M (as saporin). The combined use of the two anti‐CD30 bimAbs further increased cytotoxicity by 100‐fold, resulting in an IC50 of 1.9 × 10−13 M. A slightly less efficient improvement was obtained by combining the CD25 × sap1 bimAb with the CD30 × sap2 bimAb directed against a different toxin epitope (saporin IC50 to 7 × 10−13 M). In contrast, no synergistic effect was observed using the combination of the anti‐CD25 bimAb with the anti‐CD30 bimAb reacting with the same epitope of saporin (IC50 = 4.5 × 10−11 M). Analysis of FITC–saporin binding to L540 cells by flow cytometry demonstrated that the appropriate combinations of the two anti‐CD30/anti‐saporin bimAbs or of the anti‐CD30/anti‐saporin and anti‐CD25/anti‐saporin bimAbs had a cooperative effect on the binding of the ribosome‐inactivating protein (RIP) to the cells, when compared with single bimAbs.
Prevalence of hereditary haemochromatosis in premature atherosclerotic vascular disease
Authors: Franco  , Zago  , Trip  , Ten Cate  , Van Den Ende  , Prins  , Kastelein  , Reitsma
Published: March 2003

It has been proposed that iron accumulation may contribute to atherogenesis by increasing free radical formation and oxidative stress. Epidemiological studies in which the association of iron status with atherosclerosis was assessed raised conflicting results. To test whether genetic haemochromatosis is associated with increased atherosclerosis, we determined the prevalence of two mutations in the HFE gene related to haemochromatosis (845G → A; Cys282Tyr, and 187 C → G, His63Asp) in 265 consecutive patients with premature (<50 years of age) angiographically‐proven atherosclerotic disease (coronary and/or peripheral), and in 272 healthy controls. PCR amplification followed by RsaI (Cys282Tyr analysis) and BclI (His63Asp analysis) restriction digestion was employed to define the genotypes. The mutant Cys282Tyr allele had a frequency of 0.07 among controls and 0.04 among patients (carrier frequency of 14.0% and 8.3%, respectively). The frequency of the His63Asp mutant allele was 0.14 (28.6% of carriers) in controls and 0.11 (22.2% of carriers) in patients. Five of 265 patients (1.1%) and 9/272 controls (3.3%) were compound heterozygotes. In conclusion, a lower prevalence of the Cys 282Tyr mutation and a similar frequency of the His63Asp mutation was observed in patients with atherosclerotic disease in comparison with normal controls. These findings do not support an association between haemochromatosis and atherogenesis.
Coexpression of erythroid and megakaryocytic genes in acute erythroblastic (FAB M6) and megakaryoblastic (FAB M7) leukaemias
Authors: Linari Silvia , Vannucchi Alessandro M. , Ciolli Stefania , Leoni Franco , Caporale Roberto , Grossi Alberto , Pagliai Gabriella , Santini Valeria , Paoletti Francesco , Ferrini Pierluigi Rossi
Published: March 2003

There is evidence to suggest a close relationship between the erythroid and megakaryocytic lineages. Using RT‐PCR, we evaluated the coexpression of erythroid and megakaryocytic genes in blasts from 25 acute myeloid leukaemia (AML) cases (FAB M1–M7) and three unclassifiable leukaemias with trilineage dysplasia (trilineal AML). All FAB M6 and M7 and trilineal leukaemias expressed mRNAs for α‐globin, glycoprotein IIb (GpIIb), erythropoietin receptor (Epo‐R) and thrombopoietin receptor (c‐mpl), but not for myeloperoxidase (MPO) which in contrast was expressed in the other FAB‐subtype leukaemias. These data support the hypothesis that blasts from M7 and M6 leukaemias may derive from (or represent) a common progenitor cell with resident bipotentiality towards the megakaryocytic and erythrocytic lineages.