bluuebunny/crossref_metadata_2025 huggingface dataset:
Intestinal iron absorption studies in mouse models of iron‐overload
Authors: Raja K. B. , Simpson R. J. , Peters T. J.
Published: March 2008

Three mouse strains have been evaluated as suitable models for investigations into the pathogenesis of iron‐overload syndromes.

Mice with hereditary heterozygous α‐thalassaemia had moderately raised reticulocyte counts, but were not anaemic and showed little, if any, iron loading. In contrast, mice with homozygous β‐thalassaemia showed microcytic anaemia, reticulocytosis and splenomegaly. Iron‐loading was marked, progressive with age and mainly confined to the spleen. Liver iron‐loading increased until the age of 7–8 weeks, with no further increase over successive weeks. Although intestinal iron absorption was modestly increased due to enhanced mucosal uptake, the majority of the ‘excess’liver and spleen iron could be accounted for by re‐distribution of iron from the erythrocytic compartment.

Homozygous hypotransferrinaemic mice, with approximately 1–2% of normal plasma transferrin levels, were markedly anaemic with hypochromic microcytic erythrocytes. Intestinal iron absorption increased 3–4‐fold (predominantly due to changes in mucosal transfer), as compared to wild‐type controls and heterozygotes, and was ascertained to be a major factor causing the marked hepatic iron overload. Heterozygous hypotransferrinaemic mice, with over half normal plasma transferrin levels and a mild degree of hepatic iron loading, showed very similar characteristics to wild‐type controls. Thus, of the three models, hpx/hpx mice showed the greatest enhancement in intestinal iron absorption and net iron‐loading and provides a suitable animal model of spontaneous iron‐overload.

Comparison of iron absorption values between the models suggests that reticulocytes cannot account for the enhanced absorption seen in the hpx/hpx mice.
Splenic sepsis in sickle cell disease
Authors: Cavenagh J. D. , Joseph A. E. A. , Dilly S. , Bevan D. H.
Published: March 2008

We describe two patients with sickle cell disease (SCD) who developed infections situated in the spleen. One patient had a splenic abscess and there was strong clinical evidence for an infected splenic infarct in the second patient. SCD predisposes to splenic infection because of functional hyposplenism, defective phagocyte function and splenic infarction. Splenic infections can occur in patients who might be considered to have an absent spleen and the diagnosis of splenic abscess should be considered in individuals with SCD who present with fever and abdominal pain.
Stem cells and the microenvironment in aplastic anaemia
Authors: Gordon M. Y.
Published: March 2008

Normal blast colony‐forming cells (BI‐CFC) bind to stroma cultured in the presence of methylprednisolone (MP+) but not to MP‐ stroma. In aplastic marrow, the incidence of BI‐CFC is variable (0–4 x normal values) and there is no consistent relationship with the CFU‐GM (granulocyte‐macrophage colony‐forming cell) content. Normal stroma require MP to induce BI‐CFC binding function and form fat cells whereas MP‐ stroma grown from 4/9 aplastic patients formed fat cells and bound BI‐CFC. The 5/9 aplastic cases that did not form fat cells spontaneously also bound BI‐CFC moderately better than normal stroma. This suggests that the haemopoietic microenvironment in aplastic anaemia responds physiologically to bone marrow failure by increasing its haemopoietic support capacity.
An improved DNA‐based identification of fetuses at risk for HPA‐1a (PlA1) neonatal alloimmune thrombocytopenia
Authors: Clemenceau Sylvie , Perichon Bruno , Elion Jacques , Kaplan Cecile , Krishnamoorthy Rajagopal
Published: March 2008

A simple and reliable procedure, based on DNA amplification and HpaII mapping, is proposed for the identification of fetuses at risk for HPA‐1a (PlA1) neonatal alloimmune thrombocytopenia which could cause life‐threatening haemorrhage, even in early fetal life. This typing procedure for HPA‐1 alleles should help in deciding, very early, the therapeutic management of the fetuses at risk.
Persistence of an activating N‐RAS oncogene mutation in clonogenic progenitor cells from an acute myeloid leukaemia patient in remission
Authors: Zaheer H. A. , Bagnara M. , Gibson F. M. , Robinson G. , Rutherford T. R. , Gordon‐Smith E. C.
Published: March 2008

Summary A patient with acute myeloid leukaemia (AML) with an activating N‐RAS oncogene mutation was studied in a haemopoietic clonogenic progenitor cell assay. Individual colonies and clusters were analysed by polymerase chain reaction and oligonucleotide hybridization for the original mutation. The mutation was detected in a majority of leukaemic clusters, but also in almost half of the differentiated colonies. After chemotherapy the patient entered clinical remission. However, the mutation could still be detected in the bone marrow. Only differentiated colonies and no leukaemic clusters were grown from the remission bone marrow, but the original mutation was still detectable in almost half of the colonies.
Serum immunoreactive interleukin‐6 and C‐reactive protein levels in patients with multiple myeloma at diagnosis
Authors: Tienhaara Anri , Pulkki Kari , Mattila Kari , Irjala Kerttu , Pelliniemi Tarja‐Terttu
Published: March 2008

Summary Serum bioactive but not immunoreactive interleukin‐6 (IL‐6), and serum C‐reactive protein (CRP), have been reported to be of prognostic significance in multiple myeloma (MM). We measured serum immunoreactive IL‐6 by a sensitive enzyme‐linked immunosorbent assay in 30 MM patients at diagnosis. In 30% of the patients serum immuno‐reactive IL‐6 exceeded the upper reference limit. The concentrations of CRP and IL‐6 showed a linear association. Logarithmically transformed IL‐6, CRP and β2‐microglobulin were significant variables by univariate survival analysis: by multivariate analysis CRP was a slightly stronger prognostic factor than IL‐6 and the only one of independent prognostic significance.
Avascular necrosis of bone after allogeneic bone marrow transplantation: clinical findings, incidence and risk factors
Authors: Socié G. , Sélimi F. , Sedel L. , Frija J. , Devergie A. , Bourdeau H. Esperou , Ribaud P. , Gluckman E.
Published: March 2008

Summary. In the present study we describe the incidence, clinical course, and management of avascular necrosis of bone following allogeneic bone marrow transplantation, and identify risk factors related to its development. All patients developing avascular necrosis of bone after allogeneic bone marrow transplantation between January 1974 and September 1992 were included in the analysis and were studied using the Hôpital Saint Louis Bone Marrow Transplant Database and hospital records. 27/727 allogeneic transplant recipients developed avascular necrosis leading to an 8·1% incidence at 5 years, by product limit estimate, ranging from 5% to 11·2%. Symptoms developed 119–1747 d (median 398 d) after transplantation. In these 27 patients a total of 52 joints were affected (mean 1·92 per patient, range 1–7). The hip joint was most often affected (69% of patients). All patients had joint pain that led to diagnosis by means of standard radiographs with or without the help of technetium‐99 scans and/or magnetic resonance imaging. All but three patients received steroid therapy for acute graft‐versus‐host disease. Among 10 factors tested, three were shown to be significantly linked to an increased risk for developing avascular necrosis by multivariate analysis: male gender (relative risk (RR) 4·2, P= 0·002), age older than 16 (RR = 3·87, P= 0·004), and acute graft‐versus‐host disease requiring steroid therapy (RR = 6·30, P= 0·0002). 10 patients (37%) required joint replacement within 19 months (range 2–42) following diagnosis of avascular necrosis. In conclusion, avascular necrosis of bone is a frequent late complication of allogeneic bone marrow transplantion causing significant morbidity and requiring replacement surgery in one‐third of affected patients. In this 18‐year single‐centre survey, older age, male gender and steroid therapy given for acute graft‐versus‐host disease were shown to independently increase the risk of avascular necrosis of bone.
Plasma granulocyte‐colony stimulating factor concentrations ([G‐CSF]) in the early neonatal period
Authors: Russell A. R. Bedford , Davies E. G. , McGuigan S. , Scopes G. J. D. , Daly S. , Gordon‐Smith E. C.
Published: March 2008

Summary We studied G‐CSF concentrations ([G‐CSF]) at birth and their relationship with neutrophil count, incidence of infection, gestational age, labour, and the presence of maternal pregnancy‐induced hypertension.

Plasma [G‐CSF] were significantly elevated in babies with suspected infection and in those of hypertensive mothers, compared to healthy babies delivered by elective caesarian section (median [range] = 3101 [75‐ > 5000] pg/ml and 153 [45–857] pg/ml versus 32 [11–266] pg/ml; P<0·0001); and were unrelated to neutrophil count and gestational age. Initial high concentrations (> 100 pg/ml) declined by 7 d (P<0·0001).
A randomized trial comparing vinblastine in slow infusion and by bolus i.v. injection in idiopathic thrombocytopenic purpura: a report on 42 patients
Authors: Facon T. , Caulier M. T. , Wattel E. , Jouet J. P. , Bauters F. , Fenaux P.
Published: March 2008

Summary. Forty‐two patients with ITP were randomly allocated to vinblastine slow infusion or bolus injection. 16/21 patients (76%) receiving slow infusion of vinblastine and 14/21 patients (67%) receiving a bolus injection of vinblastine had a response. There was no superiority of slow infusions of vinblastine over intravenous bolus injections, either when recently diagnosed and chronic ITP were analysed together or separately. In our opinion, intravenous bolus injections of vinblastine might be preferred, because they are easier to perform and have a better local tolerance.
A comparison of the sensitivity of blood and bone marrow for the detection of minimal residual disease in chronic myeloid leukaemia
Authors: Lin Feng , Goldman John M. , Cross Nicholas C. P.
Published: March 2008

Summary. We have sought to determine whether peripheral blood or bone marrow is more sensitive for assessment of minimal residual disease in chronic myeloid leukaemia (CML). Contemporaneous blood and marrow specimens were taken from 21 patients at various times after allogeneic bone marrow transplant (BMT) and from one patient in complete cytogenetic remission on α‐interferon. Samples were analysed for evidence of BCR‐ABL mRNA by RT‐PCR: four were PCR negative and 19 PCR positive. Results with blood and marrow were concordant in all cases. BCR‐ABL transcripts were quantified in PCR‐positive samples using a competitive PCR titration assay. Results ranged from <10 to 2 × 106 BCR‐ABL transcripts/μg RNA. In all 19 cases a high degree of concordance in BCR‐ABL levels with blood and marrow (r=0·99) was found. We conclude that either tissue may be used for residual disease studies after BMT for CML.
The effect of rGM‐CSF on neutrophil and eosinophil regeneration after ABMT as monitored by circulating levels of granule proteins
Authors: Höglund M. , Simonsson B. , Smedmyr B. , Öberg G. , Venge P.
Published: March 2008

Summary.In order to further evaluate the effects of rGM‐CSF on the reconstituting granulopoiesis, plasma and serum levels of myeloperoxidase (MPO) and lactoferrin (LF), as well as serum levels of eosinophil cationic protein (ECP), were monitored daily during a period of 3–4 weeks following ABMT in a group of 22 patients treated with either rGM‐CSF (n=11) or placebo (n=11). Despite faster increase in the neutrophil counts in the rGM‐CSF group, we did not observe any difference either in P‐MPO or in P‐LF during the period of early engraftment (days 11–19). This finding indicates that the proliferative effect of rGM‐CSF on the neutropoiesis may be overestimated when neutrophil counts alone are taken into consideration, and suggests that other mechanisms may have contributed to the increase in the number of circulating neutrophils. The ratio of the serum to plasma level of LF, but not of MPO, was higher in the rGM‐CSF group, probably reflecting a specificin vivoneutrophil priming effect. In the rGM‐CSF group there was a clear increase of S‐ECP during the second and third week post transplant, corresponding to an increase in eosinophil counts, which indicates that rGM‐CSF stimulated eosinophil reconstitution without causing excessive activation of the mature eosinophils.
Liposomal amphotericin B in the treatment of fungal infections in neutropenic patients: a single‐centre experience of 133 episodes in 116 patients
Authors: Mills W. , Chopra R. , Linch D. C. , Goldstone A. H.
Published: March 2008

Summary.Liposomal amphotericin B (AmBisome) was used for suspected or confirmed fungal infection complicating 133 neutropenic episodes in 116 patients not tolerating, or not responding to, conventional amphotericin. Adverse effects were infrequent and no significant renal impairment resulted. Acute reactions occurred in five patients, reversible hepatic dysfunction in 23, and hypernatraemia in 17. The putative mycosis resolved with AmBisome treatment in 81 episodes (61%) and progressed with fatal outcome in 25 (19%), but the diagnosis was equivocal in most, and in 27 episodes (20%) evidence indicating nonfungal pathogenesis emerged. Treatment efficacy is, however, evaluable in those with proven aspergillosis. 13/17 patients with confirmed invasive aspergillosis responded to AmBisome (77%), conventional amphotericin having failed in 11. Treatment was successfully discontinued when the neutrophil count was <1 × 109/l in eight responders (61%). In four further patients treated for suspected aspergillosis, disseminated infection was documented at post‐mortem, but the true incidence is unknown. This analysis confirms that AmBisome is well tolerated and effective against invasive mycoses in neutropenic patients, and may salvage patients when conventional amphotericin proves excessively toxic or ineffective.
Multiplex analysis of two intragenic microsatellite repeat polymorphisms in the genetic diagnosis of haemophilia A
Authors: Windsor Sharon , Taylor Sherryl A. M. , Lillicrap David
Published: March 2008

Summary. Use of molecular genetic studies for carrier detection and prenatal diagnosis of haemophilia A will be informative in approximately 75% of kindreds with a prior history of the disorder if previously reported bi‐allelic intragenic polymorphisms are used. In this study we report the use of two multi‐allelic, microsatellite repeat polymorphisms within the factor VIII gene in genetic testing for this disease. These two, dinucleotide repeat, polymorphisms have been analysed using a multiplex polymerase chain reaction (PCR) protocol, and results can be available within 3 d of receipt of samples. At the intron 13 polymorphic locus we have confirmed the original observation of eight alleles, whilst at the intron 22 locus, in contrast to a preliminary report of this polymorphism, we have seen five alleles. We have analysed 32 families (174 subjects) with the two microsatellite repeat markers and have found that using these two polymorphisms alone, 81% of families are informative for linkage analysis. The intron 13 repeat was informative in 22/31 families tested (71%) and the intron 22 polymorphism was informative in 12/17 families (71%). We have also found that in 11/32 families tested (34%) the microsatellite repeats were the only informative intragenic markers. In view of these observations, we believe that the most effective strategy for initiating haemophilia A genetic testing is to combine the multiplex PCR analysis of the intron 13 and 22 dinucleotide repeats with the PCR analysis of the intron 18 BclI marker. In our population this protocol provided informative results in approximately 88% of families and results can be available within 3 d of receiving the samples for testing.
Molecular characterization of illegitimate TCRδ gene rearrangements in acute myeloid leukaemia
Authors: Przybylski G. , Oettle H. , Ludwig W. D. , Siegert W. , Schmidt C. A.
Published: March 2008

SUMMARY. Recently, we and others have shown the occurrence of TCRδ gene rearrangements in acute myeloid leukaemia (AML). In this study we describe the molecular characteristics of these rearrangements by the polymerase chain reaction (PCR) and the direct sequencing of PCR products. 11 rearrangements were characterized in blast cell samples from six patients. We found a heterogenous pattern of TCRδ gene rearrangements with involvement of Vδ1‐5 regions. These findings differ from observations in T‐ALL and B‐cell precursor ALL, where predominantly usage of Vδ1 and Vδ2 regions has been described. Furthermore, extensive diversity of junctional sites was observed, including addition of up to 37 N nucleotides, nucleotide deletions at junction sites of Vδ and Jδ segments and usage of up to three Dδ segments. The Dδ3 fragment was the most frequently used diversity element and was found in 10 rearrangements. Nine of the 11 rearrangements were non‐functional, either incomplete or out of the reading frame. Therefore a functional TCRδ cannot be expressed in these myeloid blast cells.
The family history and inherited thrombophilia
Authors: Briéut Ernest , van der Meer Felix J. M. , Rosendaal Frits R. , Houwing‐Duistermaat Jeanine J. , van Houwelingen Hans C.
Published: March 2008

SUMMARY. The role of the family history as a tool for the diagnosis of inherited thrombophilia has not been established. Several authors have indicated that a positive family history is not a good predictor of inherited abnormalities such as antithrombin III deficiency, or deficiencies of protein C or protein S. We have tried to approach the family history in a quantitative way. To this end we used the cumulative incidence data of thrombosis in the general population and also in a population of protein C deficient families to estimate the expected number of symptomatic subjects in a family under both the hypothesis of inherited thrombophilia and the null‐hypothesis. Although a number of assumptions underlying our calculations need to be verified and probably adjusted before any truly quantitative meaning can be assigned to this approach, we feel that the family history is a useful diagnostic test for inherited thrombophilia if it is used in a critical way.
Tumour necrosis factor‐α and interleukin 4 promote the differentiation of myeloma cell precursors in multiple myeloma
Authors: Sawamura Morio , Murakami Hirokazu , Tamura Jun'ichi , Matsushima Takafumi , Sato Sadao , Naruse Takuji , Tsuchiya Jun
Published: March 2008

Summary. The effects of tumour necrosis factor‐α (TNF‐α) and interleukin 4 (IL‐4) on peripheral blood mononuclear cells (PBMC) from 36 patients with multiple myeloma (MM), 12 with monoclonal gammopathy of undetermined significance (MGUS) and 21 normal controls, were investigated. In 16/36 patients with MM, monoclonal plasma cells appeared after 4d in cultures containing TNF‐α and IL‐4. These changes were not observed in PBMC from patients with MGUS or from normal controls. These findings suggest that myeloma cell precursors do exist in the peripheral blood of MM patients and differentiate into plasma cells in the presence of TNF‐α and IL‐4. Based on these observations, we think that the variation in the number of myeloma cell precursors in peripheral blood could be used as a prognostic parameter of response to chemotherapy in myeloma patients. In addition, this assay may be useful to distinguish early‐stage MM from MGUS.
Red blood cell aggregability in patients with a history of leg vein thrombosis: influence of post‐thrombotic treatment
Authors: Chabanel Anne , Horellou Marie Hélé;ne , Conard Jacqueline , Samama Meyer M.
Published: March 2008

Summary. Reversible aggregation or red blood cells (RBC) plays an important role in determining blood flow properties, and it is this aggregation which increases blood viscositgy at low shear rates. The structure and sites of venous thrombi, as well as the fact that stasis is a major predisposing factor in venous thrombosis, suggest a strong association between vein thrombosis, slow blood flow and increased blood viscosity. RBC aggregation and disaggregation were measured (SEFAM erythroaggregameter, France) in 54 patients with a history of unexplained leg vein thrombosis. Results were compared to those of controls classified according to age. Increased RBC aggregability was observed in 41% of the patients, and the mean values indicated a significant elevation of RBC aggregability in patients when compared with controls (P < 0.05). Subgroups were compared to study the influence of thrombus recurrence and thrombosis type (deep versus superficial vein thrombosis) on the aggregation parameters. No significant difference was found between these subgroups. The use of compression stockings and veinotropic drugs tended to reduce the abnormalities in RBC aggregability (P < 0.05). An increase in RBC aggregability and in the shear resistance of RBC aggregates, by predisposing ro circulatory stasis, is likely to contribute to the evolution and complications of leg vein thrombosis.
Detection of herpesvirus type 6 by polymerase chain reaction in blood donors: random tests and prospective longitudinal studies
Authors: Wilborn Freimut , Schmidt Christian Andreas , Zimmermann Robert , Brinkmann Volker , Neipel Frank , Siegert Wolfgang
Published: March 2008

Summary. In order to evaluate the prevalence of HHV‐6 in blood donors, we examined 112 persons by polymerase chain reaction (PCR) and ELISA. HHV‐6 antibodies could be detected in 107/111 (96.4%) of the donors. The median ELISA antibody level was 0.451 (range 0.056–0.914). 14 individuals (12.5%) were PCR positive in either oral lavage fluid, urine or buffy coat. Six persons (5.4%) were PCR positive in buffy coat samples. The prospective longitudinal analysis of 11 donors for periods between 7 and 13 weeks revealed that 4/6 persons who were initially PCR negative had positive tests in 9/63 weeks studied. Two persons were consistently PCR positive over the whole observation period of 12 and 13 weeks. HHV‐6 variants could be determined in 14 persons as variant A in nine and variant B in five cases. These observations emphasize the high prevalance of HHV‐6 and suggest that some blood donors carry detectable concentrations of the virus and therefore may be a source for transmission of HHV‐6. The finding of positive PCR in antibody negative individuals suggests that antibody determination may not be sufficient to identify potentially infectious persons.
Prenatal diagnosis of congenital dysgranulopoietic neutropenia
Authors: Koren A. , Cohen H. , Shneyour I. , Shalev E.
Published: March 2008

Summary. Congenital dysgranulopoietic neutropenia is a severe life‐threatening disease characterized by specific dysmorphic granulocytes. Prenatal diagnosis of congenital neutropenia was reported first in 1983. In 1989 we reported a family with congenital dysgranulopoietic neutropenia in two siblings (one male and one female): in 1992 the mother became pregnant and prenatal diagnosis was performed by cordocentesis. The results we obtained from the fetus at risk enabled us to suggest that it was not affected, and we advised the parents that the pregnancy could continue: a healthy female was born. The neutrophil count at 2 and 4 months was normal.
Hepatitis C virus infection in patients with non‐Hodgkin's lymphoma
Authors: Ferri Clodoveo , Caracciolo Francesco , Zignego Anna Linda , Civita Luca La , Monti Monica , Longombardo Giovanni , Lombardini Francesco , Greco Francesco , Capochiani Enrico , Mazzoni Alessandro , Mazzaro Cesare , Pasero Giampiero
Published: March 2008

Summary. Hepatitis C virus (HCV), which is both a hepatotropic and a lymphotropic virus, has been proposed as a possible causative agent of mixed cryoglobulinaemia. This ‘benign’ lymphoproliferative disorder can switch over to a malignant B‐cell non‐Hodgkin's lymphoma (NHL). Therefore HCV infection has been investigated in a series of 50 unselected Italian patients with B‐cell NHL. Antibodies against HCV were found in 30% of NHL and HCV viraemia in 32% of cases. HCV‐related markers were detected in 34% (17/50) of our NHL patients; this prevalence is particularly significant when compared with HCV seropositivity in Hodgkin's lymphoma (3%) and healthy controls (1.3%).
Recombinant human interleukin‐3: pharmacokinetics after intravenous and subcutaneous bolus injection and effects on granulocyte kinetics
Authors: Hovgaard D. J. , Folke M. , Mortensen B. T. , Nissen N. I.
Published: March 2008

The pharmacokinetics of E. coli derived recombinant human interleukin‐3 (rhIL‐3) was studied following intravenous (i.v.) and subcutaneous (s.c.) bolus injection of rhIL‐3. After i.v. bolus injection in eight patients, serum peak levels of 34.5‐135.0 ng/ml were reached, followed by a rapid decline with a t1/2α of 17 ± 2 min and a t1/2β of 59 ± 7 min. After s.c. bolus injection in five patients, the absorption was more prolonged with peak serum levels reached at 2.8 ± 0.4h. Elimination was also more protracted, and serum base‐line levels were reached at 14‐24 h.

The immediate effect of rhIL‐3 on peripheral white blood cells was less pronounced and more variable than previously found for G‐ or GM‐CSF. Following i.v. administration, neutrophils showed a moderate drop to median 64% of initial values (range 42‐85%) at median 30 min after injection (range 15‐60 min) at median 30 min after injection (range 15‐60 min) followed by an increase at 24 h to 69‐288% of initial values. Eosinophils dropped to a median nadir of 34% and then gradually increased to maximum values in the range 135‐720% at 18‐24h. The effect of rhIL‐3 was further examined following i.v. injection of autologous 111Indium‐labelled granulocytes in six patients. In steady state, i.v. injection of rhIL‐3 caused a moderate drop in 111Indium activity of peripheral blood within 20 min without tendency to subsequent recovery. No change occurred in the activity over the lungs and liver. The activity over the spleen decreased moderately in two patients. These results are strikingly different from those previously obtained after i.v. injection of rhGM‐CSF.
Bone marrow angiogenesis and progression in multiple myeloma
Authors: Vacca Angelo , Ribatti Domenico , Roncali Luisa , Ranieri Girolamo , Serio Gabriella , Silvestris Franco , Dammacco Franco
Published: March 2008

Tumour growth is angiogenesis‐dependent. We found a high correlation between the extent of bone marrow angiogenesis, evaluated as microvessel area, and the proliferating (S‐phase) fraction of marrow plasma cells, evaluated as labelling index (LI), in patients with multiple myeloma (MM) and in those with monoclonal gammopathies of undetermined significance (MGUS). Angiogenesis itself was significantly associated with active as opposed to non‐active MM and MGUS. The highest microvessel area accompanied rapidly progressive MM with the highest LI. When a cut‐off value of 2% or greater of the microvessel area was used, most patients with active MM were classified correctly. The risk of active disease in patients with MM increased in parallel with the microvessel area. A causal relationship between plasma cell growth, activity phase in MM and marrow angiogenesis is suggested. Since angiogenesis proceeds in step with the enlargement of plasma cell tumours and the activity phase in MM, its measurement could be a useful prognostic marker in patients with plasma cell proliferative disorders.
Efficacy of an analysis of lymphocyte subsets in predicting the clinical response to α‐interferon therapy in thalassaemia patients with chronic infection by hepatitis C virus: a pilot study
Authors: Russo‐Mancuso Giovanna , Gregorio Felicia Di , Passero Enrica , Sciotto Antonella , Mazzarino Maria Clorinda , Malaponte Grazia , Schilirog Gino
Published: March 2008

Summary. α‐interferon (α‐IFN) has been used to treat chronic non‐A non‐B hepatitis in thalassaemic patients with response rates from 45% to 83%. Unfortunately, treatment with α‐IFN is associated with side‐effects which have a negative effect on the quality of life of the patient. Therefore it would be useful if we could distinguish in advance those patients who would benefit from such therapy from those who would not. In the present study we found that the modification of lymphocyte subsets 20 h after the administration of the first dose of α‐IFN revealed that relative numbers of T helper lymphocytes (CD4+) increased in three non‐responding patients and decreased in five responding patients, whereas those of T suppressor lymphocytes (CD8+), and natural killer cells (CD57+. CD16+) decreased in non‐responding patients and increased in responding patients. Therefore analysis of the lymphocyte subsets CD4, CD8, CD57 and CD16 before and 20 h after the administration of α‐IFN can be used to predict the clinical response to treatment with α‐IFN.
The SCID mouse as a model for multiple myeloma
Authors: Ahsmann Els J. M. , Tol Maarten J. D. van , Oudeman‐Gruber Joy , Lokhorst Henk , Uytdehaag Fons G. C. M. , Schuurman Henk‐Jan , Bloem Andries C.
Published: March 2008

Summary. The SCID mouse was investigated as a potential animal model for human multiple myeloma (MM). Duplicate samples of bone marrow mononuclear cells (BMMC) and/or peripheral blood mononuclear cells (PBMC) of six MM patients in different clinical phases and one patient with monoclonal gammapathy of undetermined significance (MGUS) were injected intraperitoneally into SCID mice. Human immunoglobulins (Ig) in the SCID sera were quantified with a light‐chain isotype‐specific ELISA, and their monoclonality biochemically characterized, using a sensitive immunoblotting technique after agar gel electrophoresis. Successful transplantation of bone marrow derived‐tumour cells in SCID mice was obtained with BMCC of two MM patients with progressive disease. Human plasma cells were detected in the mesenteric fat tissue around the pancreas and the spleen. This model in SCID mice may facilitate studies on processes involved in tumour progression and provides a new tool for therapeutic approaches in MM.
A flow cytometric assay using mepacrine for study of uptake and release of platelet dense granule contents
Authors: Wall Judith E. , Buijs‐Wilts Marleen , Arnold Julie T. , Wang Winfred , White Melanie M. , Jennings Lisa K. , Jackson Carl W.
Published: March 2008

Summary. Diagnosis of platelet dense granule storage pool disease and release defects at present requires a combination of studies including lumiaggregometry, conventional platelet aggregation, radioactive serotonin uptake and release, and electron microscopy. Flow cytometric methods have been developed to study platelet activation, aggregation, and α‐granule protein release. Here, we have investigated the use of flow cytometry for analysis of platelet dense granule content uptake and release using mepacrine as a fluorescent marker. Mepacrine (quinacrine) is rapidly taken up and localized in dense granules of platelets. For the assay, as little as 20 μl of blood from a fingerstick collected without anticoagulant or venous blood collected in 3.8% sodium citrate were diluted 1:40 with 2 ml Hanks balanced salt solution (BSS). 300 μl of this cell suspension were incubated with mepacrinc alone, or simultaneously with a mouse monoclonal antibody to human platelet glycoprotein IIb (Tab), used as a platelet‐specific marker. The bound monoclonal antibody was then indirectly labelled with the fluorochrome, RED670. 100 μl of the sample were further diluted with Hanks BSS for one‐ or two‐colour flow cytometric analysis. To verify that mepacrine uptake was related to platelet dense granule content, platelets of beige mice, a strain with dense granule deficiency, were examined. Their mepacrine uptake was substantially decreased compared to that of normal mice. Decreased mepacrine uptake also was demonstrated in platelets of a patient with Hermansky‐Pudlak syndrome in which a deficiency of platelet dense granules is characteristic. In both human and mouse platelets, mepacrine uptake was proportional to platelet size. Thrombin induced mepacrine release in a dose‐dependent manner from 0.003 to 0.4 U/ml. Therefore both platelet uptake and release of mepacrine can be readily detected by flow cytometry. Flow cytometry provides an attractive alternative to aggregation and radioactive serotonin as methods to study defects in platelet dense granule function.
Rapid and sensitive non‐radioactive assay for the detection of clonal gene rearrangements in B‐lineage acute leukaemia
Authors: Martin Gerald , Mairs Robert , Platt Jeremy , Gibson Brenda
Published: March 2008

Summary. We have applied a simple and non‐radioactive assay system used in conjunction with polymerase chain reaction amplification and high‐resolution polyacrylamide gel electrophoresis to detect clonal immunoglobulin gene rearrangements in leukaemia patient DNA. This technique is as sensitive as isotopic methods, allowing the detection of clonal DNA diluted 1 in 10 000 in non‐clonal DNA.
Low incidence of MDR1 expression in acute promyelocytic leukaemia
Authors: DRACH DORIS , ZHAO SHOURONG , DRACH JOHANNES , ANDREEFF MICHAEL
Published: March 2008

Summary. The MDR1 gene product, P‐glycoprotein, functions as a transmembrane efflux pump for certain cytotoxic agents including anthracyclines. Based upon the clinical observation that patients with acute promyelocytic leukaemia (APL) respond favourably to anthracyclines, we hypothesized that APL cells may have low levels of MDR1 expression. We therefore investigated MDR1 expression in 10 patients with APL and compared results with those obtained in 18 patients with other subtypes of acute myelogenous leukaemia (AML). Prior to reverse transcriptase polymerase chain reaction with MDR1 specific primers, leukaemic cells were purified by fluorescence activated cell sorting to exclude normal haemopoietic cells, in particular lymphocytes, from the MDR1 analysis. In sorted APL cells, MDR1 expression was detected in only two of 10 patients, which was significantly different from findings in other AML subtypes (MDR1 expression in 14/18 patients; P<0.01). When unsorted specimens from APL patients were studied, five of six cases were MDR1 positive, whereas sorted APL cells were shown to express MDR1 mRNA in only one of these cases. MDR1 mRNA levels expressed as MDRl/beta‐2 microglobulin ratios were significantly lower in APL (0‐24 –0‐2, mean –SD) than in AML (0‐75 –0‐48; P<0.01). We conclude that low or absent expression of MDR1 in APL cells may contribute to the efficacy of anthracyclines in the treatment of APL.
Probability of progenitor renewal (PPR) and production of clonogenic progeny (CFU‐GM) by primitive adherent progenitor cells in adult human bone marrow and umbilical cord blood
Authors: GORDON M. Y. , LEWIS J. L. , SCOTT M. A. , ROBERTS I. A. G. , GOLDMAN J. M.
Published: March 2008

Summary. Human haemopoietic tissues contain primitive plastic‐adherent progenitor cells (PΔ cells) that can be detected by measurement of their granulocyte‐macrophage colony‐forming cell (CFU‐GM) progeny. Limiting dilution analysis and Poisson statistics are necessary for determining the frequency of PΔ cells because each of them produces several CFU‐GM. Limiting dilution also permits measurement of the abilities of individual PΔ progenitors to produce CFU‐GM. Here we report that the frequencies of PΔ progenitors in cord blood and adult marrow are similar (5.6 and 7.8/105 mononuclear cells respectively) and individual cord blood PΔ progenitors produce fewer CFU‐GM than adult PΔ progenitors. To test the possibility that the lower production of differentiated progeny by cord blood cells was the result of a higher rate of self‐renewal, we devised a two‐stage limiting dilution assay relying on the relative production of CFU‐GM after two consecutive weeks of incubation. The probability of progenitor renewal (PPR) was derived from the number of wells (progenitors) that produced CFU‐GM on both occasions compared with the number that produced CFU‐GM on the first occasion only. The total number of CFU‐GM produced on the second occasion compared with the number produced on the first occasion provided an index of the overall change in the size of the PΔ cell population. The data indicate that PΔ cells in cord blood have a higher PPR (0.59) than those in adult marrow (036). Also, the relative numbers of CFU‐GM produced in the second and first weeks were greater for cord blood (1.2) than for adult marrow (0.36). Therefore PΔ cells in cord blood have a greater capacity for self‐maintenance and possibly for expansion than PΔ cells in adult marrow.
In vivo effects of interleukin‐11 and stem cell factor in combination with erythropoietin in the regulation of erythropoiesis
Authors: HAAN GKRALD DE , DONTJE BERT , ENGEL CHRISTOPH , LOEFFLER MARKUS , NIJHOF WILLEM
Published: March 2008

Summary. In this study we evaluated the in vivo effects of interleukin‐11 (IL‐11) and stem cell factor (SCF), in combination with erythropoietin (EPO) on murine erythropoiesis. Mice were treated for 7d with IL‐11, SCF and EPO, each at three dose levels. In total, 27 different dose combinations were tested. IL‐11 as well as SCF could only marginally stimulate erythroid progenitor cell numbers, but IL‐11 in combination with SCF was able to increase BFU‐E and CFU‐E numbers 4‐fold, in the absence of exogenous EPO. This resulted in an increased reticulocyte count. In contrast with the stimulatory effect on immature erythroid cell stages, IL‐11 treatment induced a mild anaemia, which probably resulted from a plasma volume expansion. The additional treatment with EPO resulted in strong synergistic effects on CFU‐E numbers. The combination of high‐dose IL‐11 and high‐dose SCF was able to increase the overall efficiency of EPO‐induced erythroid amplification, which was reflected by a left‐shift of the in vivo EPO dose‐response curve. The stimulating effects of IL‐11 and SCF were further demonstrated when the effects on the reticulocyte count of a single high‐dose EPO injection were assessed in normal and SCF + IL‐11 treated mice. Whereas a single EPO dose increased the reticulocyte count by a factor of 3, IL‐11 + SCF pretreatment increased this to a factor of 7. This study shows that in vivo SCF and IL‐11 are important modulators of red blood cell production. First, these factors probably increase the input from the stem cell compartment into the erythroid lineage, where subsequently EPO is required for further amplification. Additionally, however, IL‐11 and SCF increase the overall efficiency of EPO‐induced amplification, probably due to a stimulatory effect on late‐stage erythroid cells and to a redistribution of cells from marrow to spleen.
Idiopathic macrocytic anaemia in the aged: molecular and cytogenetic findings
Authors: ANTTILA PEKKA , IHALAINEN JARKKO , SALO ARMI , HEISKANEN MERVI , JUVONEN EEVA , PALOTIE AARNO
Published: March 2008

Summary. Macrocytosis in the elderly is often caused by abnormalities of haematological stem cell differentiation. In this study, a group of elderly patients was analysed for four molecular and cell biological parameters. The aim of the study was to screen elderly patients with idiopathic macrocytic anaemia or MDS for a set of alterations which are related to haematological dysplasia. The analyses used were: DNA‐methylation at the calcitonin A gene 5′‐area, NBAS point mutations at codons 12 and 13, in vitro colony formation of peripheral blood progenitor cells and cytogenetics of bone marrow cells. The results show that a significant portion of elderly patients with idiopathic macrocytosis have one or more of the abnormalities analysed. Hypermethylation of the calcitonin A gene 5′‐area at the chromosome 11 band pi 5 is relatively common (7/15). Chromosomal aberrations (3/12) and NRAS oncogene point mutations (0/15) were rare findings. In vitro culture of erythroid progenitor cells was relatively frequently abnormal (7/15). Eight of our nine macrocytic patients who did not fulfill the FAB criteria for MDS had at least one of the alterations studied; this suggests that these patients might represent early phases of a stem cell disorder.
BCL‐2 expression by leukaemic blasts in a SCID mouse model of biphenotypic leukaemia associated with the t(4;ll)(q21;q23) translocation
Authors: POCOCK C. F. E. , MALONE M. , BOOTH M. , EVANS M. , MORGAN G. , GREIL J. , COTTER F. E.
Published: March 2008

Summary. Acute leukaemia of infancy is associated with abnormalities at chromosome band llq23, and has a poor prognosis. The gene involved, Mixed Lineage Leukaemia (MLL), has been identified and has the characteristics of a transcription factor. The BCL‐2 gene responsible for blocking of programmed cell death is highly expressed in a number of haematological malignancies, both with and without the t(14;18) translocation. Those without the translocation include acute lymphoblastic leukaemia (ALL), acute myeloid leukaemia (AML) and chronic lymphocytic leukaemia (CLL). In these diseases the BCL‐2 protein is implicated in drug resistance to apoptosis‐inducing chemotherapeutic agents. High BCL‐2 expression is also associated with autonomous growth of leukaemic blasts in culture and predicts a poor prognosis. The SEM cell line, established using blood lymphoblasts from a 5‐year‐old girl in first relapse with t(4;l 1) ALL, expresses lymphoid (CD19) and myeloid (CD13) cell surface markers. In cell culture, a subpopulation of cells (<30%) express the BCL‐2 protein. A reproducible model of true biphenotypic leukaemia in the SCID mouse has been established using the SEM‐K2 cell line (a subclone of the SEM cell line). Between 5 and 50 million cells injected intravenously (i.v.) produce complete replacement of the murine bone marrow by day 30, associated with blood lymphoblastosis and infiltration of the spleen. No tumour masses were seen. Fluorescence in situ hybridization (FISH) analysis of the cell line and blood from the SCID‐human (SCID‐hu) chimaera has confirmed the presence of the t(4;ll). Reverse transcriptional‐polymerase chain reaction (RT‐PCR) reveals that the breakpoint lies between exons 7 and 8 of the MLL‐1 gene on chromosome 11 (the main breakpoint region). A further translocation, t(7;13), has been identified. Fluorescent antibody cell sorter (FACS) analysis of tumour material recovered from the SCID‐hu model confirms expression of CD19 and CD13 identical to that of the cell line. In addition, BCL‐2 expression in SCID‐hu marrow is now seen in the majority of tumour cells. BCL‐2 expression appears to confer a survival advantage to the blast cells in vivo. This reproducible model of biphenotypic leukaemia suggests that BCL‐2 expression may play a role in leukaemogenesis. The model is suitable for the investigation of gene‐targeted therapy, including antisense oligonucleotides, directed towards the MLL and BCL‐2 genes.
Binding of cardiolipin to polystyrene beads: evidence for a lamellar phase orientation
Authors: STEWART MICHAEL W. , GORDON PHILIP A. , ETCHES WAI S. , MARUSYK HALYNA , POPPEMA SIBRAND , BIGAMI COLIN , SYKESI BRIAN
Published: March 2008

Summary. The association of cardiolipin with polystyrene beads was studied using 31P‐NMR and electron microscopy. In the presence and absence of fetal calf serum, cardiolipin appeared to bind to the polystyrene beads in lamellar phase as assessed by 31P‐NMR imaging. Electron microscopic analysis revealed an even coating of phospholipid about the beads with extensive micelle binding. Cardiolipin‐coated beads challenged with ACA‐positive sera followed by immunogold indicated antibody bound to micelles associated with the bead. Studies conducted with ACA IgG purified from patient sera indicated that some ACA bound to CL beads in the absence of a source of ACA cofactor (i.e. gelatin‐blocked beads), some ACA required β2‐GPI for binding (i.e. no binding in the presence of β2‐GPI‐depleted plasma), whereas other ACA which showed negliglible binding with gelatin‐blocked beads, showed enhanced binding in the presence of /?2‐GPI‐depleted plasma. The data indicate that: (1) cardiolipin binds to polystyrene beads in lamellar phase, (2) ACA bind to phospholipid micelles bound directly to the polystyrene beads, and (3) ACA differ between individuals displaying varying phospholipid and phospholipid/cofactor substrate specificities.
Modulation of all‐trans retinoid acid pharmacokinetics in acute promyelocytic leukaemia by prolonged interferon‐α therapy
Authors: LAZZARINO MARIO , CORSO ALESSANDRO , REGAZZI MARIO B , IACONA ISABELLA , BERNASCONI CARLO
Published: March 2008

Summary. Continuous treatment with all‐trans retinoid acid (ATRA) induces accelerated drag catabolism which is considered responsible for acquired resistance to ATRA. We studied the effect of interferon‐o:2a (IFN) on ATRA pharmacokinetics in two patients with acute promyelocytic leukaemia (APL) in complete remission maintained by alternating 15d of IFN and 15 d of ATRA. Day 15 ATRA levels obtained during IFN + ATRA treatment were significantly higher than those observed in patients maintained on ATRA alone. In one patient IFN was discontinued and day 15 ATRA levels decreased to those observed in patients scheduled for maintenance with ATRA alone. In our two patients IFN substantially reduced the induction of ATRA catabolism, indicating a potential role for IFN in modulating ATRA pharmacokinetics.
Validation of a model to predict survival in elderly patients with acute myeloid leukaemia
Authors: JOHNSON PETER R. E. , RYDER W. DAVID J. , LIU YIN JOHN A.
Published: March 2008

Summary.Intensive chemotherapy is the treatment of choice for selected elderly patients with acute myeloid leukaemia (AML). We recently developed a model to predict survival, thereby providing objective information upon which to select appropriate therapy for such patients. Such models, however, must be validated on a cohort of patients not used during the development of the model.

We have tested the model using a series of 61 elderly patients consecutively treated with intensive chemotherapy.

Using several statistical techniques, we have shown that the model is of value in predicting prognosis, though two patients did markedly better than the model prediction. This model may be useful for predicting survival in elderly patients with AML and warrants more extensive validation.