bluuebunny/crossref_metadata_2025 huggingface dataset:
Cardiovascular effects of melatonin in hypertensive patients well controlled by nifedipine: a 24‐hour study
Authors: Lusardi Paola , Piazza Elena , Fogari Roberto
Published: March 2003

Aims
As melatonin has been found to play a role in the mechanisms of cardiovascular regulation, we designed the present study to evaluate whether the evening ingestion of the pineal hormone might interfere with the antihypertensive therapy in hypertensive patients well‐controlled by nifedipine monotherapy.

Methods
Forty‐seven mild to moderate essential hypertensive outpatients taking nifedipine GITS 30 or 60 mg monotherapy at 08.30 h for at least 3 months, were given placebo or melatonin 5 mg at 22.30 h for 4 weeks according to a double‐blind cross‐over study. At the end of each treatment period patients underwent a 24 h noninvasive ambulatory blood pressure monitoring (ABPM) during usual working days; sleeping period was scheduled to last from 23.00 to 07.00 h.

Results
The evening administration of melatonin induced an increase of blood pressure and heart rate throughout the 24 h period (ΔSBP = + 6.5 mmHg, P < 0.001; ΔDBP = + 4.9 mmHg, P < 0.01; ΔHR = + 3.9 beats min−1, P < 0.01). The DBP as well as the HR increase were particularly evident during the morning and the afternoon hours.

Conclusions
We hypothesize that competition between melatonin and nifedipine, is able to impair the antihypertensive efficacy of the calcium channel blocker. This suggests caution in uncontrolled use of melatonin in hypertensive patients. As the pineal hormone might interfere with calcium channel blocker therapy, it cannot be considered simply a dietary supplement.
Compliance with tricyclic antidepressants: the value of four different methods of assessment
Authors: George C. F. , Peveler R. C. , Heiliger S. , Thompson C.
Published: March 2003

Aims
To assess the advantages and disadvantages of four methods for studying compliance with antidepressants: self‐report scores, tablet counts, a microprocessor (MEMS) container system and the assay of nordothiepin and dothiepin concentrations in plasma.

Methods
The techniques were used in 88 patients commencing tricyclic antidepressants in the setting of UK general practice.

Results
The MEMS system proved to be the most informative technique allowing identification of the precise time of container opening, the demonstration of ‘drug holidays’ and early cessation of therapy. Self‐report scores (Morisky) proved a useful screening technique with a sensitivity of 72.2% and specificity of 74.1% for ≥ 80% compliance. Although tablet counts were possible in 84 patients (95.5%) they were unreliable in 19 (21.6%). Blood concentration assays proved the least acceptable method to patients and were possible in only 53 (60.2%). A ratio of nordothiepin:dothiepin ≥ 1.1 claimed, by others, to identify noncompliance was only reliable when concentrations were low.

Conclusions
Both the MEMS system and self‐report scores proved useful methods for identifying noncompliant patients in the setting of UK general practice. Although compliance was higher than reported in previous studies with 70 patients (79.5%) completing 6 weeks treatment, general practitioners tended to prescribe subtherapeutic doses.
Pharmacokinetic interactions between sildenafil and saquinavir/ritonavir
Authors: Muirhead Gary J. , Wulff Maria B. , Fielding Anitra , Kleinermans Diane , Buss Neil
Published: March 2003

Aims
To investigate the effect of the antiretroviral protease inhibitors saquinavir (soft gelatin capsule) and ritonavir on the pharmacokinetic properties and tolerability of sildenafil and to investigate the effect of sildenafil on the steady‐state pharmacokinetics of saquinavir and ritonavir.

Methods
Two independent, 8 day, open, randomized, placebo‐controlled, parallel‐group studies (containing a double‐blind crossover phase) were conducted at Pfizer Clinical research units (Canterbury, UK. and Brussels, Belgium). Twenty‐eight healthy male volunteers entered each study. In each study, volunteers were randomized (n = 14 per group) to receive sildenafil on day 1 followed by a 7‐day treatment period (days 2–8) with saquinavir or placebo (Study I) or ritonavir or placebo (Study II). Sildenafil or placebo (Study I and Study II) was administered alternately on day 7 or day 8, depending on initial randomization. The effect of saquinavir and ritonavir on the pharmacokinetics of sildenafil and its primary circulating metabolite (UK‐103, 320) and the effect of single‐dose sildenafil on the steady‐state pharmacokinetics of saquinavir (1200 mg three times daily) and ritonavir (500 mg twice daily) were determined. The safety and tolerability of sildenafil coadministered with saquinavir or ritonavir were also assessed.

Results
Both protease inhibitors significantly increased Cmax, AUC, tmax and t½ values for both sildenafil and UK‐103, 320. Ritonavir showed a significantly greater effect than saquinavir with increases in sildenafil AUC and Cmax of 11‐fold (95% CI: 9.0, 12.0) and 3.9‐fold (95% CI: 3.2, 4.9), respectively. This compared with increases of 3.1‐fold (95% CI: 2.5, 4.0) and 2.4‐fold (95% CI: 1.8, 3.3) for coadministration with saquinavir. In contrast, the steady‐state pharmacokinetics of saquinavir and ritonavir were unaffected by sildenafil. The increases in systemic exposure to sildenafil and UK‐103, 320 were not associated with an increased incidence of adverse events or clinically significant changes in blood pressure, heart rate or ECG parameters.

Conclusions
These results indicate that both saquinavir and ritonavir modify the pharmacokinetics of sildenafil presumably through inhibition of CYP3A4. The more pronounced effect of ritonavir may be attributed to its additional potent inhibition of CYP2C9. No change in safety or tolerability was observed when sildenafil was coadministered with either protease inhibitor. However, given the extent of the interactions, a lower sildenafil starting dose (25 mg) should be considered for patients receiving saquinavir and it is recommended not to exceed a maximum single dose of 25 mg in a 48 h period for patients receiving ritonavir.
Lack of effect of grapefruit juice on the pharmacokinetics and pharmacodynamics of amlodipine
Authors: Vincent John , Harris Stuart I. , Foulds George , Dogolo Lisa C. , Willavize Susan , Friedman Hylar L.
Published: March 2003

Aims
To determine whether repeated once daily administration of grapefruit juice altered the pharmacokinetics or pharmacodynamics of the calcium antagonist amlodipine.

Methods
The effects of grapefruit juice on the pharmacokinetics and pharmacodynamics of oral and intravenous amlodipine were assessed in 20 healthy men in a placebo‐controlled, open, randomized, four‐way crossover study using single doses of amlodipine 10 mg. For 9 days beginning with the day of administration of amlodipine, grapefruit juice (or water control) was given once daily, and blood samples, blood pressure and heart rate measures were obtained. Plasma concentrations of amlodipine and its enantiomers were determined in separate assays by GC‐ECD.

Results
Oral amlodipine had high systemic availability (grapefruit juice: 88%; water: 81%). Pharmacokinetic parameters of racemic amlodipine (AUC, Cmax, tmax, and kel) were not markedly changed with grapefruit juice coadministration. Total plasma clearance and volume of distribution, calculated after intravenous amlodipine, were essentially unchanged by grapefruit juice (CL 6.65 ml min−1 kg−1, juice vs 6.93 ml min−1 kg−1, water; Vdss 22.7 l kg−1, juice vs 21.0 l kg−1, water). Grapefruit juice coadministration did not greatly alter the stereoselectivity in amlodipine oral or intravenous kinetics. The sum of S(–) and R(+) enantiomer concentrations correlated well with total racemic amlodipine concentration (r2 = 0.957; P = 0.0001). Coadministration of grapefruit juice with either route of amlodipine administration did not significantly alter blood pressure changes vs control.

Conclusions
Grapefruit juice has no appreciable effect on amlodipine pharmacodynamics or pharmacokinetics, including its stereoselective kinetics. Bioavailability enhancement by grapefruit juice, noted with other dihydropyridine calcium antagonists, does not occur with amlodipine. Once daily grapefruit juice administration with usual oral doses of amlodipine is unlikely to alter the profile of response in clinical practice.
Clinical risk factors for venous thromboembolus in users of the combined oral contraceptive pill
Authors: Black Corri , Kaye James A. , Jick Hershel
Published: March 2003

Aims  To estimate the risk of venous thromboembolism among women prescribed the oral contraceptive pill who have acute clinical conditions such as lower limb fractures, compared with women with idiopathic venous thromboembolism.

Methods  A nested case‐control analysis using the General Practice Research Database, January 1993 to December 1999 was carried out. The participants were women aged 15–39 years, prescribed third generation oral contraceptives (gestodene and desogestrel) or oral contraceptives containing levonorgestrel. The main outcome measures were odds ratios as a measure of the relative risk estimate for venous thromboembolism in women with clinical conditions that predispose to VTE.

Results  The adjusted relative risk estimate for venous thromboembolism among patients with the acute clinical conditions, compared with those without such illness, and adjusted for oral contraceptive use, was 17 (95% CI 6.5, 46).

Conclusions  This paper documents the strong independent association between certain acute clinical conditions and venous thromboembolism in women prescribed oral contraceptives. Failure to accurately identify and exclude such patients from a study of the effect of oral contraceptives on the risk of venous thromboembolism would result in an underestimate of the risk of venous thromboembolism associated with oral contraceptives.
Oxidation of celecoxib by polymorphic cytochrome P450 2C9 and alcohol dehydrogenase
Authors: Sandberg Mia , Yasar Ümit , Strömberg Patrik , Höög Jan‐Olov , Eliasson Erik
Published: March 2003

Aims
Celecoxib is a novel selective cyclooxygenase‐2 inhibitor, which is subject to extensive hepatic metabolism. The aims of the present in vitro investigation were 1) to compare the rate of celecoxib hydroxylation by different genetic variants of cytochrome P450 2C9 (CYP2C9), and 2) to identify the enzyme(s) involved in the formation of the major metabolite carboxycelecoxib.

Methods
Hydroxycelecoxib formation was studied in human liver microsomes from 35 genotyped livers, as well as in yeast microsomes with recombinant expression of different P450 variants. Carboxycelecoxib formation was studied in liver microsomes incubated in the absence or presence of liver cytosol. The metabolites were identified and quantified by h.p.l.c. In addition, hydroxycelecoxib oxidation by different variants of recombinant human alcohol dehydrogenase (ADH1–3) was analysed by spectrophotometric monitoring of NADH generation from NAD+.

Results
 The  intrinsic  clearance  of  celecoxib  hydroxylation  was  significantly  lower for yeast‐expressed CYP2C9.3 (0.14 ml min−1 nmol−1 enzyme) compared with CYP2C9.1  (0.44 ml min−1 nmol−1 enzyme).  In  human  liver  microsomes,  a  signifi­cant 2‐fold decrease in the rate of hydroxycelecoxib formation was evident in CYP2C9*1/*3 samples compared with CYP2C9*1/*1 samples. There was also a marked reduction (up to 5.3 times) of hydroxycelecoxib formation in a liver sample genotyped as CYP2C9*3/*3. However, the CYP2C9*2 samples did not differ significantly from CYP2C9*1 in any of the systems studied. Inhibition experiments with sulphaphenazole (SPZ) or triacetyloleandomycin indicated that celecoxib hydroxylation in human liver microsomes was mainly dependent on CYP2C9 and not CYP3A4. The further oxidation of hydroxycelecoxib to carboxycelecoxib was completely dependent on liver cytosol and NAD+. Additional experiments showed that ADH1 and ADH2 catalysed this reaction in vitro with apparent Km values of 42 µm and 10 µm, respectively, whereas ADH3 showed no activity.

Conclusions
The results confirm that CYP2C9 is the major enzyme for celecoxib hydroxylation in vitro and further indicate that the CYP2C9*3 allelic variant is associated with markedly slower metabolism. Furthermore, it was shown for the first time that carboxycelecoxib formation is dependent on cytosolic alcohol dehydrogenase, presumably ADH1 and/or ADH2.
Quinine pharmacokinetics in chronic haemodialysis patients
Authors: Roy Louise ,   Martine Leblanc,, Bannon Pierre , Villeneuve Jean‐Pierre
Published: March 2003

Aims Quinine is often used to prevent muscle cramps in patients with chronic renal failure. A standard dose of 300 mg at bedtime is usually recommended, but little is known about the pharmacokinetics of quinine in the presence of renal failure.

Methods We studied the pharmacokinetics of quinine in eight normal subjects and eight patients with chronic renal failure on haemodialysis after a single oral dose of quinine sulphate (300 mg).

Results The concentration of α1‐acid glycoprotein (AAG), the major binding protein for quinine, was increased in haemodialysis patients compared with control subjects (1.52 g l−1vs 0.63 g l−1[mean difference 1.033; 95% CI 0.735, 1.330]) whereas albumin levels were decreased (30 g l−1vs 40 g l−1[mean difference 9.5; 95% CI 3.048, 15.952]). Accordingly, the free fraction of quinine was decreased (0.024 vs 0.063 [mean difference 0.0380; 95% CI 0.0221, 0.0539]) and the apparent volume of distribution tended to decrease (0.95 l kg−1vs 1.43 l kg−1[mean difference 0.480; 95% CI 0.193, 1.154]). The quinine binding ratio correlated with the plasma concentration of AAG but not that of albumin. The clearance of free (unbound) quinine was increased in haemodialysis patients compared with controls (67.9 ml min−1 kg−1vs 41.1 ml min−1 kg−1[mean difference −26.8; 95% CI, –56.994, 3.469]), and the area under the curve (AUC) of the two main metabolites, 3‐hydroxyquinine and 10,11‐dihydroxydihydroquinine were increased.

Conclusions In patients with chronic renal failure, there is an increase in plasma protein binding and in the clearance of free drug, resulting in lower plasma concentration of free quinine.
REVIEWCytokines and chemokines in the initiation and regulation of epidermal Langerhans cell mobilization
Authors: Kimber I. , Cumberbatch M. , Dearman R.J. , Bhushan M. , Griffiths C.E.M.
Published: March 2003

Langerhans cells (LC) are members of the wider family of dendritic cells. LC reside in the epidermis where they serve as sentinels of the immune system, their responsibilities being to sample the external environment for changes and challenges and to deliver information (antigen) to responsive T lymphocytes within skin draining lymph nodes. The ability of LC to migrate from the epidermis to regional lymph nodes is therefore of pivotal importance to the induction of cutaneous immune responses. The journey that LC have to make from the skin has a number of requirements. Initially it is necessary that LC disassociate themselves from surrounding keratinocytes and are liberated from other influences that encourage their retention in the epidermis. Subsequently, migrating LC must successfully traverse the basement membrane of the dermal–epidermal junction and make their way, via afferent lymphatics, to draining lymph nodes. Effective entry into lymph nodes is necessary, as is correct positioning of cells within the paracortex. There is increasing evidence that both cytokines and chemokines, and their interaction with appropriate receptors expressed by LC, orchestrate the mobilization and movement of these cells. We here consider the parts played by these molecules, and how collectively they induce and direct LC migration.
Antineutrophil cytoplasmic antibodies and HLA class II alleles in minocycline-induced lupus-like syndrome
Authors: Dunphy J. , Oliver M. , Rands A.L. , Lovell C.R. , Mchugh N.J.
Published: March 2003

We report 14 patients with minocycline-induced lupus-like syndrome (four men, 10 women; mean age 27·8 years) who developed a lupus-like illness after chronic use of minocycline for acne (1–10 years, median 3·8). Clinical features resolved completely on drug withdrawal (mean follow-up 11 months) and reappeared in two patients who were rechallenged. Sera from all 14 patients contained antineutrophil cytoplasmic antibodies (ANCA) giving a perinuclear pattern on indirect immunofluorescence on ethanol-fixed human neutrophils (p-ANCA), whereas 14 control asymptomatic individuals taking minocycline for acne were ANCA-negative. Eleven of the 14 patients had elevated antimyeloperoxidase antibodies and 10 had antielastase antibodies on enzyme-linked immunosorbent assay, which diminished on extended follow-up, as did other serological abnormalities. Major histocompatibility complex class II typing demonstrated that all of the 13 patients tested were either HLA-DR4 (nine of 13) or HLA-DR2 (four of 13) positive, and all had an HLA-DQB1 allele encoding for tyrosine at position 30 of the first domain. Our findings suggest a model whereby the presence of p-ANCA may be a marker for the development of lupus-like symptoms in genetically susceptible individuals taking minocycline for acne.
The clinical spectrum of epidermolysis bullosa simplex
Authors: Horn H.M. , Tidman M.J.
Published: March 2003

As part of the U.K. National Epidermolysis Bullosa Register, we have systematically recorded clinical information on 130 (77%) of the 168 known Scottish epidermolysis bullosa simplex (EBS) sufferers. Three subtypes of EBS were recognized: Dowling–Meara (EBS-DM), Weber–Cockayne (EBS-WC) and Köbner (EBS-Kb), seen in 5%, 42% and 53% of patients, respectively. As there is considerable overlap between EBS-WC and EBS-Kb, with both phenotypes frequently seen within the same pedigree, EBS-WC is best regarded as a milder variant of EBS-Kb rather than a separate disorder. Improvement with age is common in all variants of EBS, but is not invariable. Pain due to acral blistering in EBS-Kb/EBS-WC has a more marked impact on life-style than the blisters of EBS-DM. Oral blistering, nail involvement and aplasia cutis congenita occur in all EBS subtypes and laryngeal involvement is a feature of EBS-DM. Seasonal variation is not seen in EBS-DM but is common in EBS-Kb/EBS-WC.
Acquired immune deficiency syndrome-related hyperkeratotic Kaposi’s sarcoma with severe lymphoedema: report of five cases
Authors: Hengge U.R. , Stocks K. , Goos M.
Published: March 2003

Kaposi’s sarcoma (KS) is the most frequent neoplasm in acquired immune deficiency syndrome (AIDS) patients. Whereas typical cases present as erythematous, plaque or nodular lesions, hyperkeratotic variants of AIDS-associated KS are rare. We describe five patients with AIDS-associated KS with hyperkeratosis and lymphoedema as prominent features. We also speculate on its pathogenesis.
Epidermolysis bullosa acquisita with ultraviolet radiationsensitivity
Authors: Jappe U. , Zillikens D. , Bonnekoh B. , Gollnick H.
Published: March 2003

A 37-year-old male patient developed a bullous eruption and erythematous plaques mainly in exposed areas following prolonged sun exposure. In addition, blisters were noted on oral and nasal mucous membranes. Histopathological examination of a lesional skin biopsy revealed a subepidermal blister. Linear deposition of IgG and C3 at the epidermal basement membrane zone was revealed by direct immunofluorescence microscopy of a perilesional skin biopsy. Indirect immunofluorescence on 1 mol/L salt-split skin showed binding of autoantibodies to the dermal side of the split. Immunoblot analysis of dermal extracts demonstrated that the patient’s serum contained IgG antibodies against type VII collagen, whereas no reaction was seen with epidermal extracts or by enzyme-linked immunosorbent assay using a recombinant form of bullous pemphigoid 180. Standardized ultraviolet (UV) radiation provocation induced blistering with both UVA (13·5 J/cm2) and UVB (0·04 J/cm2) within 24 h clinically and histologically. External and systemic UV-protective medication and nine cycles of high dosage immunoglobulins given intravenously (1·2 g/kg body weight over 2–3 days every 4 weeks) resulted in the cessation of blister formation. To the best of our knowledge, this is the first report of a case of epidermolysis bullosa acquisita with sensitivity to UV.
Diminished production of interleukin‐6 in chronic lymphocytic leukaemia (B‐CLL) cells from patients at advanced stages of disease
Authors: Hulkkonen Janne , Vilpo Juhani , Vilpo Leena , Hurme Mikko ,   for the Tampere CLL Group
Published: March 2003

The production of the cytokines interleukin‐6 (IL‐6) and tumour necrosis factor‐alpha (TNF‐α) in B‐CLL cells from 24 patients at different stages of chronic lymphocytic B‐cell leukaemia (B‐CLL) was investigated in vitro. In the majority of these cases, low spontaneous IL‐6 production was measured. Mitogenic stimulation with phorbol 12‐myristate 13‐acetate (PMA) or PMA plus interleukin‐2 (IL‐2) resulted in a tremendous increase in TNF‐α and IL‐6 production in cells representing early stage (Binet A) disease. In contrast, very little, if any, production took place in cells from patients with advanced stage (Binet C) B‐CLL. The results from stage B patients were intermediate. The most remarkable difference was recorded in PMA‐stimulated (1 ng/ml) IL‐6 production. In stimulated 72 h cultures, IL‐6 concentrations were 1280 ± 1080 pg/ml for Binet A (n = 11), 757 ± 597 pg/ml for Binet B (n = 8) and 46.0 ± 84.0 pg/ml for Binet C (n = 5). The differences in IL‐6 production between stage C v B and stage C v A were both statistically significant (P = 0.025). Similar effects, but to a lesser extent, were observed in TNF‐α production. These results suggest that the varying capacity to produce IL‐6 and TNF‐α may play a role in B‐CLL progression and in clinical manifestations of the disease.
Binding and regulation of thrombopoietin to human megakaryocytes
Authors: Sato Takeyuki , Fuse Akira , Niimi Hiroo , Fielder Paul J. , Avraham Hava
Published: March 2003

Thrombopoietin (TPO, c‐Mpl ligand) is considered to play an important role in the regulation of megakaryocytopoiesis and platelet production by activating the cytokine receptor c‐Mpl. We have examined the binding of 125I‐TPO to the human megakaryocytic cell line, CMK, and to primary human megakaryocytes. Scatchard analysis of TPO binding to its cognate receptor in megakaryocytic cells suggested the existence of a single class of c‐Mpl receptors. CMK cells exhibited 1223 receptors per cell with a dissociation constant (Kd) of Kd = 223 p
M, whereas primary human megakaryocytes exhibited 12 140 receptors per cell and a dissociation constant of Kd = 749 p
M. The pretreatment of CMK cells and primary bone marrow megakaryocytes with TPO resulted in a decreased binding of TPO to the c‐Mpl receptors. This down‐regulation was observed within 3 h and was not inhibited by cycloheximide. Phorbol ester, an activator of protein kinase C, also inhibited TPO binding to the c‐Mpl receptors by reducing the number of these receptors. The pretreatment of CMK cells with IL‐3, IL‐6 and DMSO, all of which induced the differentiation of CMK cells, did not affect the binding of TPO to the c‐Mpl receptors. These results suggest an additional mechanism, where protein kinase C may help to regulate the binding of TPO to these cells.
Cost‐effectiveness and quality‐of‐life assessment of GM‐CSF as an adjunct to intensive remission induction chemotherapy in elderly patients with acute myeloid leukaemia
Authors: Uyl‐DE Groot  , LÖwenberg  , Vellenga  , Suciu  , Willemze  , Rutten
Published: March 2003

We conducted a prospective, randomized, multicentre clinical trial comparing the effects and costs of GM‐CSF as an adjunct to intensive chemotherapy in elderly patients with acute myeloid leukaemia (AML). The patients were randomized to either daunomycin–cytosine arabinoside (control arm: n = 161) or daunomycin–cytosine arabinoside with GM‐CSF (GM‐CSF arm: n = 157). The primary end‐point was the effect of GM‐CSF on the percentage of complete remissions (CR). Survival duration, disease‐free survival, quality of life and costs were evaluated separately.

CR after remission induction treatment was achieved in 55% of the patients in the control group and in 56% of the patients in the GM‐CSF group (P = NS). The duration of survival and disease‐free survival at 2 years after randomization were estimated at 22% and 19% for the control group and 22% and 14% for the GM‐CSF group (P = NS). Considering the short‐term quality of life, the administration of GM‐CSF resulted in more problems with regard to depressed mood, diarrhoea and rash/eczema. With regard to the long‐term quality of life there were no significant differences between the two groups. The average costs of the primary treatment were higher in GM‐CSF‐treated patients than in the control group, i.e. US$40782 and US$34465, respectively (P < 0.01). The costs during the follow‐up period did not differ between the two groups.

The results of this randomized clinical trial indicate that daunomycin–cytosine arabinoside plus GM‐CSF is not a cost‐effective treatment strategy when compared with daunomycin–cytosine arabinoside alone.
Functional analysis of the p53 protein in AIDS‐related non‐Hodgkin's lymphomas and polymorphic lymphoproliferations
Authors: Martin Antoine , Flaman Jean‐Michel , Frebourg Thierry , Davi FRÉDÉric , EL Mansouri Said , Amouroux Jacques , RaphaËL Martine
Published: March 2003

Alteration of the tumour suppressor gene p53 is frequent in AIDS‐related non‐Hodgkin's lymphomas (AIDS‐NHL), particularly in Burkitt's or Burkitt's‐like lymphomas (BL/BLL). Since mechanisms of inactivation other than mutations have been advanced, the transcriptional activity of the p53 protein was studied in a functional assay in yeast in a series of AIDS‐NHL lesions and compared with their morphology, immunohistochemistry (IHC) and single‐strand conformation polymorphism (SSCP) analysis detection of other p53 abnormalities, Epstein‐Barr virus (EBV) status, MDM‐2 oncoprotein expression and c‐MYC rearrangement. Polymorphic lymphoproliferations (PL), identified as precursors of NHL in HIV‐patients, were also analysed in attempt to detect p53 modifications related to clonal progression.

The functional assay detected p53 mutants in 40% (12/30) of the tumours: 50% (6/12) of BL/BLL, 40% (4/10) of diffuse large cell lymphomas (DLCL) and 25% (2/8) of PL. An oligoclonal or monoclonal population was identified in the two PL cases with mutant p53. An accumulation of the p53 protein was detected by IHC in 26% (8/30) of the tumours (five BL/BLL and three DLCL) and was associated with positive functional assay. In the 20 lesions tested by both of the screening methods for mutations, a p53 mutant pattern was detected in 55% of cases (11/20) and in 25% of cases (5/20) respectively with the functional assay and SSCP analysis of exons 5–8. There was no inverse correlation between the detection of EBV genome and the presence of p53 mutations and no overexpression of MDM‐2 protein for the whole series. In conclusion, the functional assay was more sensitive than IHC and SSCP for the detection of p53 mutations in tumour samples. The mutations identified in AIDS‐NHL lesions inactivate the p53 protein and in PL they could represent a selection of an aggressive clone.
Evidence that the ubiquitin proteolytic pathway is involved in the degradation of precipitated globin chains in thalassaemia
Authors: Wickramasinghe  , Lee
Published: March 2003

Ultrastructural immunocytochemical studies were performed on sections of bone marrow from three patients with β‐thalassaemia major and two patients with haemoglobin H (HbH) disease. Some sections were reacted with either a polyclonal or a monoclonal anti‐human‐ubiquitin antibody and the reaction visualized using a gold‐labelled secondary antibody. The inclusions of precipitated globin chains found within the erythropoietic cells of all five patients reacted much more strongly than the surrounding inclusion‐free cytoplasm with both of the anti‐ubiquitin antibodies, indicating that the precipitated globin chains were ubiquitinated. A non‐specific reaction between the anti‐ubiquitin antibodies and the inclusions was excluded by demonstrating that various other antibodies, including a polyclonal anti‐human cathepsin D antibody, did not react with the inclusions. The data suggest that the ubiquitin proteolytic pathway is involved in the degradation of precipitated globin chains in α‐ and β‐thalassaemia.
A warfarin induction regimen for out‐patient anticoagulation in patients with atrial fibrillation
Authors: Tait  , Sefcick
Published: March 2003

Currently available protocols for induction of warfarin anticoagulation employ initial doses of 10 mg and are best suited to in‐patient use. However, with the increasing number of elderly patients with atrial fibrillation requiring anticoagulation, there is a need for a less intense regimen which could be used for out‐patients. We have established such a regimen and report on its prospective evaluation in 37 patients referred for out‐patient initiation of warfarin, and a non‐randomized comparison with 37 in‐patients, with similar diagnoses, commenced on a traditional warfarin protocol. After exclusion of five patients on amiodarone, all of whom experienced supratherapeutic International Normalized Ratio (INR) results, the new out‐patient regimen, employing an initial 5 mg dose, resulted in a lower maximum INR during the first 21 d therapy (median 2.9 v 4.0; P = 0.0001) and fewer INRs > 4.5 (2/36 v 9/33) compared to the traditional 10 mg regimen. Time to reach stable anticoagulation was similar with each regimen; however, the 5 mg regimen gave a more accurate prediction of maintenance dose (correlation coefficient for predicted versus actual maintenance dose, r = 0.985). In comparison to a traditional 10 mg protocol, the proposed 5 mg warfarin induction regimen proved both safer and more reliable for initiation of prophylactic anticoagulation in patients with atrial fibrillation.
The dose of granulocyte colony‐stimulating factor administered following cytotoxic chemotherapy is not related to the rebound level of circulating CD34+ haemopoietic progenitor cells during marrow recovery
Authors: Martin‐Murea  , Voso  , Hohaus  , PFÖrsich  , Fruehauf  , Goldschmidt  , Hegenbart  , Haas
Published: March 2003

We report on the RmetHuG‐CSF (filgrastim)‐related mobilization efficiency in 120 patients with multiple myeloma who received cytotoxic chemotherapy. Three schedules of G‐CSF administration starting 24 h after the end of chemotherapy were used: (1) a standard dose of 300 μg/d until the completion of PBSC collection; (2) dose escalation from 300 to 600–1200 μg/d during marrow recovery; (3) 600 or 1200 μg/d starting 24 h after cytotoxic chemotherapy. As a result, the individual dose per kg bodyweight varied between 2.83 and 23.08 μg. No relationship was found between the dose of G‐CSF administered and the peak level of circulating CD34+ cells or the CD34+ cell counts recorded over the entire collection period.
Specific depletion of alloreactive T cells in HLA‐identical siblings: a method for separating graft‐versus‐host and graft‐versus‐leukaemia reactions
Authors: Mavroudis  , Dermime  , Molldrem  , Jiang  , Raptis  , Van Rhee  , Hensel  , Fellowes  , Eliopoulos  , Barrett
Published: March 2003

Accumulating evidence indicates that alloreactive donor T cells confer both graft‐versus‐host (GVH) and graft‐versus‐leukaemia (GVL) reactivity following allogeneic bone marrow transplantation. We have developed a method to deplete alloreactive donor T cells with an immunotoxin targeting the α chain of the IL‐2 receptor. In patients with chronic myeloid leukaemia and their HLA‐identical sibling donors, we measured donor helper T‐lymphocyte precursor frequencies (HTLPf) against recipient peripheral blood mononuclear cells (PBMNC; donor versus host), recipient leukaemia cells (donor versus leukaemia) and third‐party PBMNC, before and after the depletion. In seven pairs there was a 4.3‐fold reduction of donor‐versus‐host HTLPf (P = 0.017), without a significant change in the donor frequencies against third party (P = 0.96). In eight further donor–recipient pairs, immunotoxin‐depleted donor versus patient PBMNC HTLPf 4.5‐fold (mean 1/155 000 before and 1/839 000 after depletion, P = 0.007). There was a smaller non‐significant 1.8‐fold reduction in donor‐versus‐leukaemia HTLPf from 1/192 000 to 1/334 000 (P = 0.19). These results suggest that selective T‐cell depletion can significantly deplete donor anti‐host reactivity while conserving antileukaemia reactivity in HLA‐matched donor–recipient pairs.
Quality assessment of CD34+ stem cell enumeration: experience of the United Kingdom National External Quality Assessment Scheme (UK NEQAS) using a unique stable whole blood preparation
Authors: Barnett  , Granger  , Storie  , Peel  , Pollitt  , Smart  , Reilly
Published: March 2003

CD34+ peripheral blood stem cell (PBSC) mobilization and harvesting has rapidly replaced autologous bone marrow as a source of stem cells for transplantation. Timing and adequacy of harvests rely upon the accurate enumeration of circulating CD34+ cells. However, previous EQA programmes have reported interlaboratory CVs as high as 284%, suggesting the need for greater standardization. In addition the routine use of fresh and/or frozen cells as analytes also introduces antigen instability as a variable factor. To circumvent this problem and achieve a true reflection of interlaboratory variation, we have used a novel whole blood preparation in which the antigenic profiles of PBSCs, as determined by flow cytometry, are retained for > 200 d. This international scheme, currently the largest in the world, distributes aliquots of stabilized whole blood bi‐monthly to 91 laboratories in 20 countries (44 U.K., 47 overseas). Participants are required to determine the percentage and absolute values for CD34+ PBSCs using in‐house techniques. Adopting such a preparation, a more accurate determination of interlaboratory variation has been possible when compared to previous EQA studies, with CVs as low as 22% and 24% for percentage and absolute counts. In addition the programme has established that a wide range of methods are in routine use, emphasizing the urgent requirement for national/international consensus guidelines.
Soluble vascular cell adhesion molecule‐1 (sVCAM‐1) is an independent prognostic marker in Hodgkin's disease
Authors: Christiansen Ilse , SundstrÖm Christer , Enblad Gunilla , Tötterman Thomas H.
Published: March 2003

Serum levels of soluble vascular cell adhesion molecule‐1 (sVCAM‐1, sCD106) were significantly elevated in patients with Hodgkin's disease (HD) (n = 101) compared to controls (n = 31) (P < 0.0001). sVCAM‐1 correlated with histology, stage, B‐symptoms, and prognostic markers (sICAM‐1, sCD30, sIL‐2R, LDH). sVCAM‐1, sICAM‐1 and sCD30 added independent prognostic information for both disease‐free and overall survival. 14 biopsies from 13 patients with HD were immunostained for VCAM‐1 and ICAM‐1. The vascular endothelium stained positive for VCAM‐1 in 10/12 evaluable biopsies and for ICAM‐1 in all evaluable biopsies. A stromal expression of both adhesion molecules precluded a precise evaluation of HRS‐cells. This led us to investigate VCAM‐1 (and ICAM‐1) expression in six Hodgkin cell lines (HDLM‐2, L428, L540, L591, DEV, KM‐H2). Two cell lines stained positive for VCAM‐1 (HDLM‐2, L591). All cell lines stained positive for ICAM‐1. sVCAM‐1 is a new prognostic marker in HD; its predictive power equals or surpasses that of sCD30 and sICAM‐1. Furthermore, two Hodgkin cell lines stained positive for VCAM‐1. This indicates that VCAM‐1 may be expressed by some HD tumour cells in vivo.
Budd‐Chiari syndrome, portal vein and mesenteric vein thrombosis in a patient homozygous for factor V Leiden mutation treated by TIPS and thrombolysis
Authors: Leebeek  , Lameris  , Van Buuren  , Gomez  , Madretsma  , Sonneveld
Published: March 2003

We present the first case of Budd‐Chiari syndrome in association with portal and mesenteric vein thrombosis in a patient homozygous for the factor V Leiden mutation. She was treated by transjugular intrahepatic portosystemic stent (TIPS) placement followed by local thrombolytic therapy. Venous outflow from the liver was established and the thrombi in the portal and mesenteric veins were lysed completely. This therapeutic approach may be used for such patients with this severe thrombotic event, who generally have a poor prognosis.
Serum levels of p55 and p75 soluble TNF receptors in adult acute leukaemia at diagnosis: correlation with clinical and biological features and outcome
Authors: Vinante Fabrizio , Rigo Antonella , Tecchio Cristina , Morosato Lorella , Nadali Gianpaolo , Ricetti Maria Maddalena , Krampera Mauro , Zanolin Elisabetta , Locatelli Francesca , Gallati Harald , Chilosi Marco , Pizzolo Giovanni
Published: March 2003

The tumour necrosis factor (TNF)/TNF‐receptor (TNFR) complex plays a role in the growth of leukaemic cells. We retrospectively investigated the relationship between pre‐treatment serum concentration of soluble TNFR (p55‐ and p75‐sTNFRs) and outcome in adult acute myeloid (AML 82 cases) and lymphoid (ALL 44 cases) leukaemia. Both sTNFRs were significantly higher in AML (p55‐sTNFR 4.53 ± 3.7, median 3.75; p75‐sTNFR 6.51 ± 5.25 ng/ml, median 4.72) and ALL sera (3.31 ± 1.5, median 2.95; 5.30 ± 2.3 ng/ml, median 4.56, respectively) than in controls (1.89 ± 0.5, median 1.98; 2.22 ± 0.8 ng/ml, median 2.37) (P < 0.01 for both sTNFRs). Fresh leukaemic cells expressed p55‐ and p75‐sTNFRs, which were modulated and released into the supernatant (SN) following short‐term in vitro culture, suggesting that in vivo sTNFRs were also leukaemia‐derived. Whereas no correlation was observed between sTNFRs and outcome in ALL, in AML higher p55‐sTNFR levels (> 3.75 ng/ml) were associated with shorter disease‐free survival (DFS) (P = 0.006) and overall survival (OS) (P = 0.0004). At multivariate analysis p55‐sTNFR was the most significant predictor of DFS (P = 0.006) and OS (P < 0.001). Our data suggest that the prognostic significance of p55‐sTNFR in AML could be related to relevant biological features of AML blasts.
Alpha thalassaemia is associated with increased soluble transferrin receptor levels
Authors: Rees  , Williams  , Maitland  , Clegg  , Weatherall
Published: March 2003

Although α+ thalassaemia is the commonest haemoglobinopathy in the world, it is not known if it is associated with significant ineffective erythropoiesis, a fact of importance in interpreting its complex interaction with malaria. To study this problem, we have measured the concentrations of soluble transferrin receptor (sTfR) and ferritin in 181 children from Vanuatu with heterozygous (68) and homozygous (46) α+ thalassaemia, and normal controls (67). sTfR concentrations were significantly higher in both homozygotes (mean 3.1 mg/l, range 2.8–3.4) and heterozygotes (2.86 mg/l, 2.6–3.2) compared to the normal controls (2.48 mg/l, 2.3–2.7), suggesting that although globin chain imbalance is minimal, there is ineffective erythropoiesis in both these conditions. Age was also shown to significantly affect sTfR, with peak levels occurring in the 5–9 years age group. Ferritin concentrations showed a similar trend, being higher in the thalassaemic groups, although this did not reach statistical significance. No individuals had low ferritin concentrations, although two had significantly elevated sTfR levels. These observations suggest that the α+ thalassaemia phenotype includes an expansion of the erythron, and may suggest possible mechanisms for the increased susceptibility in babies with α thalassaemia to both P. falciparum and P. vivax malaria.
Mutation of p16, p21 or cyclin dependent kinase 4 is rare in acute lymphoblastic leukaemia
Authors: Qari Mohammed H. , Khalil Salim H. , Kambouris Marios , Meyer Brian F.
Published: March 2003

Homozygous deletion of the p16 tumour suppressor gene (at frequencies ranging from 14% to 29%) have been implicated in the pathogenesis of acute lymphoblastic leukaemia (ALL) by several studies. We investigated the prevalence of this deletion in a group of 46 Arab patients with common ALL. Deletion of p16 was assessed in a multiplex PCR which amplified a 405 bp fragment from exon 2 of the p16 gene, and a 242 bp fragment of the ApoE lipoprotein gene which served as an internal control. Homozygous deletion of p16 in tumour cells could be readily detected in samples containing >75% blasts. Surprisingly, none of the cases in our study showed homozygous deletion of the p16 gene. We also investigated the possibility of other genetic alterations in the p16 gene or mutation in the p21 and CDK4 (not previously reported in ALL) genes which are part of the same signal transduction pathway. A heterozygous G → A transition at nucleotide position 273 of the p16 gene was present in one patient, but did not result in an amino acid change. A C → A transversion at codon 88 of the p21 gene, which results in replacement of a phenylalanine with a leucine at position 63, was detected in one patient. In another patient a G → C transversion in exon 2 at codon 82 (5′‐untranslated region of the CDK4 gene) was detected. Results of this study showed mutation of p16, p21 or CDK4 to be rare events in Arab ALL patients.
Plasminogen Kanagawa‐I, a novel missense mutation, is caused by the amino acid substitution G732R
Authors: Higuchi Yumiko , Furihata Kenichi , Ueno Ichiro , Ishikawa Shinsuke , Okumura Nobuo , Tozuka Minoru , Sakurai Noriko
Published: March 2003

A new dysplasminogen, plasminogen Kanagawa‐I, was identified in a healthy male with no previous thrombotic episodes. His plasma plasminogen (PLG) activity was 51.4% of that of normal pooled plasma (reference interval 70–130%) and the antigen level was 94.2% of that of normal pooled plasma (reference interval 80–150%). Nucleotide sequencing revealed a heterozygous G to A transition in exon 18, which resulted in an amino acid substitution of G732R. Both the proband's father and paternal grandfather were heterozygous for this mutation. Interestingly, the grandfather was found to be a compound heterozygote for plasminogen Kanagawa‐I and Tochigi (A601T), so that his plasminogen activity and antigen level was 7.7% and 87.2% of that of normal pooled plasma, respectively. However, he has never been affected by significant thrombosis.
Laboratory abnormalities in thrombotic thrombocytopenic purpura
Authors: Rock  , Kelton John G. , Shumak Kenneth H. , Buskard Noel A. , Sutton David M. C. , Barrett Benny  ,   Members of the Canadian Apheresis Group
Published: March 2003

Thrombotic thrombocytopenic purpura is an uncommon disorder that requires prompt recognition and intervention to prevent death. To date, information regarding the classic laboratory abnormalities in the disease has been derived from small numbers of patients whose laboratory tests have been done at many different sites. We report the laboratory findings in 135 patients who presented with thrombotic thrombocytopenic purpura to 17 Canadian centres. 50 men and 85 women had a mean platelet count of 25.3 ± 19.4 × 109/l. The initial platelet count correlated with mortality; 32% of patients with a platelet count of 20 × 109/l or less died compared with 18% of patients with a platelet count >20 × 109/l (P = 0.058). The platelet‐associated IgG was elevated in 88% at presentation whereas the indirect platelet suspension immunofluorescence test was positive in only 18%. 93% of the sera showed reactivity against platelets following protein blotting. All sera tested also showed reactivity against endothelial cells. Immune complexes were seen in all patients, whereas the platelet aggregating factor was detected in 59%. Although the von Willebrand factor was elevated in the majority of patients at entry, the multimer pattern was variable and showed no predictive pattern. Renal dysfunction was common (18%).
The difference in NK‐cell activity between patients with non‐Hodgkin's lymphomas and Hodgkin's disease
Authors: Konjević Gordana , Jurišić Vladimir , Banićević Božidar , Spužić Ivan
Published: March 2003

Natural killer (NK) cells play an important role in immune surveillance against malignant diseases. Considering the lymphoid origin of malignant lymphomas, as well as scarce data concerning NK‐cell function in these neoplasms, we evaluated NK‐cell activity in 49 patients with non‐Hodgkin's lymphomas (NHL) and 47 patients with Hodgkin's disease (HD), prior to therapy. Using the recommended International Working Formulation and the Ann Arbor staging system for classification of lymphomas we found, by the LDH release cytotoxicity assay, that decreased NK‐cell activity (P < 0.05) in NHL patients was essentially related to unfavourable histology (13 indolent lymphomas, 25 intermediate and 11 very aggressive lymphomas were included), but that within these categories clinical stage of the disease also contributed to the degree of NK‐cell dysfunction. In contrast, in HD, NK‐cell activity was persistently decreased (P < 0.05), compared to controls, irrespective of histological type and clinical stage. It is of interest also that the most profound NK‐cell dysfunction that is present and persistent from the onset of HD, and which appears in very aggressive NHL was associated with the phenomenon of increased spontaneous lactate acid dehydrogenase (LDH) release activity from the separated PBMC of these patients. The difference in the level of NK‐cell impairment between patients with various histological grades of malignancy in NHL and HD suggests different initial participation of innate immune reactions in these diseases.
The role of 2‐chlorodeoxyadenosine in the treatment of patients with refractory angioimmunoblastic lymphadenopathy with dysproteinemia
Authors: Sallah Sabah , Wehbie Robert , Lepera Pamela , Sallah Waddah , Bobzien William
Published: March 2003

Treatment of patients with angioimmunoblastic lymphadenopathy with dysproteinaemia (AILD) often constitutes a challenge for the clinical haematologist. Single‐ agent and combination chemotherapy have failed to increase the response rate or survival of patients with AILD. A total of seven patients with refractory or relapsed AILD were treated with 2‐chlorodeoxyadenosine (2‐CdA) for variable number of cycles. The overall response rate was 57% with two patients (28.5%) achieving complete and sustained response. 2‐Chlorodeoxyadenosine appears to be an active agent for patients with previously treated AILD. Phase II studies evaluating the efficacy of this agent as front‐line treatment for AILD are justified, especially in the absence of any effective therapy for this disorder.