bluuebunny/crossref_metadata_2025 huggingface dataset:
B‐cell chronic lymphocytic leukaemia with CD8 expression: report of 10 cases and immunochemical analysis of the CD8 antigen
Authors: Mulligan  , Dao  , Francis  , Thomas  , Gibson  , Cole‐Sinclair  , Wolf
Published: March 2003

We report 10 cases of B‐cell chronic lymphocytic leukaemia (B‐CLL) with expression of the T‐cell antigen CD8. The majority of patients had typical B‐cell CLL with stable and non‐progressive stage A(O) disease except for more common expression of lambda light chain and CD25. Two patients had progressive disease and required therapy, one with atypical morphological and phenotypic features. The incidence of CD8 expression was approximately 0.5% of B‐CLL patients from our institutions. Immunoprecipitation of the CD8 antigen from four of these B‐CLLs showed identity to the CD8 antigen expressed on T cells with precipitation of CD8α bands of molecular weight ~34 kD. In view of the known intracellular signalling mechanism of CD8 using the tyrosine kinase p56‐lck, we studied p56‐lck expression by Western blot and found lack of consistent expression of the CD8 surface antigen, with most lacking p56‐lck. Our report indicates that CD8 expression in B‐CLL is probably under‐recognized but is not a marker of disease progression. The CD8 on the B‐CLL surface is immunochemically identical to the antigen on T cells, but is not accompanied by its usual signalling mechanism of p56‐lck tyrosine kinase and therefore is unlikely to be a functionally active receptor.
Gamma‐irradiation does not impair ATRA‐induced maturation of myeloid leukaemic cells: implication for combined radiation and differentiation therapy
Authors: Neildez‐Nguyen Thi My Anh , Chapel Alain , Arock Michel , Vétillard Jérôme , Thierry Dominique
Published: March 2003

In the present study we investigated the effects of various doses of γ‐irradiation, followed by induction of granulocytic differentiation with all‐trans‐retinoic acid (ATRA), on proliferative rate, differentiation capability and oxidative metabolism of leukaemic cells from two different myeloid leukaemia cell lines, HL‐60 and PLB‐985.

Regarding the effects of such combined treatment on the proliferative capabilities of HL‐60 and PLB‐985 cell lines, we showed that their growth kinetics were similar after 2 Gy γ‐irradiation combined with ATRA. However, with doses >2 Gy, the behaviour of the cell lines differed largely. Indeed, HL‐60 appeared to be more radiosensitive than PLB‐985 regarding cell viability and proliferation. Besides, whatever dose of irradiation (2, 5 or 10 Gy) was applied, ATRA was still able to induce differentiation of HL‐60 and PLB‐985 into granulocytes that retained the capacity to produce superoxide anion.

The results of these in vitro studies suggest that leukaemia cell lines retain their ability to respond to ATRA, a granulocytic‐differentiating inducer following high doses of irradiation. This may have implications for the use of radiation therapy in combination with ATRA for the treat‐ment of extramedullary infiltrations of myeloid leukaemias in humans.
Major surgery seems not to influence HIV disease progression in haemophilia patients
Authors: Astermark Jan , Löfqvist Thomas , Schulman Sam , Stigendal Lennart , Lethagen Stefan , Nilsson Inga Marie , Berntorp Erik
Published: March 2003

The influence of major surgery on HIV disease progression and decline in CD4+ cell count was evaluated in 23 seropositive haemophilia patients. 24 HIV‐infected patients served as non‐operated controls. In addition, 32 age‐matched seronegative subjects were included. The follow‐up time was up to 5 years.

During the course of the study, eight of the operated (35%) and 11 of the non‐operated (48%) subjects developed HIV‐related symptoms (P = 0.267). The relative risk for developing HIV‐related symptoms after surgery was 0.60 (95% CI 0.25; 1.48). A significant decline in CD4+ cell counts was observed in both the surgery (4.0 × 106/l/month, 95% CI 2.0; 6.0 × 106, P = 0.001) and the non‐surgery (4.0 × 106/l/month, 95% CI 2.0; 6.0 × 106, P = 0.004) seropositive subgroup, but no difference between the two subgroups was seen (P = 0.793). HIV (6.0 × 106/l/month, 95% CI 2.1; 9.9 × 106, P = 0.0005) but not surgery (−1.0 × 106/l/month, 95% CI −3.0; 0.96 × 106, P = 0.647) was an independent predictor for the decline in CD34+ cell count. No interaction effect was observed between HIV infection and surgery (P = 0.361). The annual amount of factor concentrate used for regular replacement therapy did not influence the decline in CD4+ cell count (P = 0.492).

We conclude that major surgery may be considered in symptom‐free HIV‐seropositive haemophilia patients, with CD4+ cell counts 0.20 × 109/l under similar premises as for seronegative subjects.
Quantitative and qualitative alterations of long‐term culture‐initiating cells in patients with acute leukaemia in complete remission
Authors: Yao  , Bouchet  , Harnois  , Giarratana  , Laporte  , Gorin  , Opez  , Douay
Published: March 2003

Bone marrow (BM) samples from 24 patients with acute leukaemia (AML 17, ALL seven) in first complete remission were compared to samples from 10 normal donors with regard to their content in long‐term culture‐initiating cells (LTC‐IC) as assessed by a limiting dilution assay and the clonogeneic capacity of these cells, in order to determine whether remission marrow cells displayed any specific defect at the primitive stem cell level. The frequency of LTC‐IC in the whole patient group was 1 in 3487 ± 3125 mononuclear cells (MNC) as compared to 1 in 794 ± 492 MNC in normal controls (P = 0.0009), with no difference between AML and ALL. Moreover, the clonogeneic capacities were 2.66 ± 0.7 (range 1.8–1.6) and 4.0 ± 1.6 (range 2.2–7.9) CFC per LTC‐IC in patients and controls respectively (P = 0.0015). These quantitative and qualitative defects were aggravated by treatment with mafosfamide at a dose of 50 μg/107 MNC/ml, where the mean recovery of LTC‐IC after in vitro purging was 42%. In nine patients autografted with purged marrow following high‐dose radiochemotherapy, no correlation could be detected between the dose of LTC‐IC (mean 6742 ± 7877/kg) and the kinetics of recovery of haemopoiesis. We concluded that, in acute leukaemia patients in complete remission, the presumably normal residual stem cell pool was not only quantitatively diminished but also qualitatively altered in its capacity to give rise to clonogeneic progenitor cells.
Analysis of mutations at the DNA repair genes in acute childhood leukaemia
Authors: Lin Ying‐Wei , Kubota Masaru , Koishi Seiji , Sawada Machiko , Usami Ikuya , Watanabe Ken‐Ichiro , Akiyama Yuichi
Published: March 2003

Deficiency in DNA repair capability is considered to be responsible for oncogenesis. Hereditary and sporadic cancers in various tissues have been reported to have mutations at the DNA repair genes. In this study we analysed two excision repair genes (ERCC1 and XPCC) and two mismatch repair genes (hMSH2 and hMTH1) in the leukaemic blasts of newly diagnosed paediatric patients by use of reverse transcriptase (RT)–polymerase chain reaction (PCR). Analysis of the leukaemic blasts from 15 patients demonstrated no alterations at the XPCC, hMSH2 or hMTH1 genes. Blasts from one patient with acute mixed lineage leukaemia revealed an abnormally migrated product of the ERCC1 gene by RT‐PCR. His leukaemic blasts showed a reduced expression of ERCC1 protein by Western blotting. Since bone marrow cells at remission showed only normally migrated product, the ERCC1 gene mutation was considered to be specific for the leukaemic blasts. This is the first report describing a mutation at the ERCC1 gene in acute childhood leukaemia.
Pregnancy and severe aplastic anaemia: causal relation or coincidence?
Authors: Oosterkamp  , Brand  , Kluin‐Nelemans  , Vandenbroucke
Published: March 2003

The relationship between aplastic anaemia (AA) and pregnancy remains uncertain. To assess whether an association between pregnancy and severe aplastic anaemia (SAA) exists, we compared the frequency of pregnancy in 35 young women with newly diagnosed SAA with the expected frequency in the general population. The observed pregnancy rate in the SAA group was 3–6%. This percentage approximates the expected pregnancy rate of 4.4% in the general population and is not compatible with a strong association between pregnancy and SAA.
Human myeloma cell apoptosis induced by interferon‐α
Authors: Otsuki Takemi , Yamada Osamu , Sakaguchi Haruko , Tomokuni Akiko , Wada Hideho , Yawata Yoshihito , Ueki Ayako
Published: March 2003

Although there have been reports regarding the clinical effectiveness of IFNα in the treatment of myeloma patients during this decade, its biological effects on human myeloma cells have still not been clarified. Recently, apoptosis has been considered as one of the most important mechanisms in the programmed cell death of malignant tumour cells induced by chemotherapeutic agents or cytotoxic immunological defence in malignancy‐carrying hosts. Among the several pathways which function to induce apoptosis, Fas and the Fas ligand system have been thought to play an important role in inducing tumour‐cell apoptosis, particularly in immunological prevention. In this study we investigated myeloma cell apoptosis induced by IFNα using five human myeloma cell lines which were established without any additional supplementation of IL‐6. In addition, the mRNA expression levels of apoptosis‐related genes employing the reverse transcriptase‐polymerase chain reaction (RT‐PCR) were also analysed with the KMS‐12‐PE cell line, which was the most sensitive of the five cell lines in terms of apoptosis induced by IFNα. Based on the results, it was determined that IFNα induced myeloma cell apoptosis in a dose‐dependent manner, but the sensitivity to IFNα in the cell lines examined varied and one cell line revealed growth stimulation by IFNα. In addition, the apoptosis induced by IFNα did not seem to be mediated by the Fas/Fas ligand pathway. Finally, the IL‐6, IL‐6R, IRF1 and IRF2 genes were up‐regulated in KMS‐12‐PE cells cultured with IFNα. Therefore these genes may play an important role during apoptosis induced by IFNα.
Inhibition of platelet activation by the Alzheimer's disease amyloid precursor protein
Authors: Henry Anna , LI Qiao‐Xin , Galatis Denise , Hesse Lars , Multhaup Gerd , Beyreuther Konrad , Masters Colin L. , Cappai Roberto
Published: March 2003

The amyloid precursor protein (APP) of Alzheimer's disease is abundantly expressed in the platelet α‐granule where its role remains unclear. This study describes a novel function for APP in regulating human platelet activation. Preincubation of platelet‐rich plasma with recombinant secreted APP (sAPP) isoforms dose‐dependently inhibited platelet aggregation and secretion induced by ADP or adrenaline. Similarly, sAPP potently inhibited low‐dose thrombin‐induced activation in washed platelet suspensions, indicating that the activity does not require plasma cofactors. There were no functional differences between sAPP forms with or without the Kunitz protease inhibitor domain or derived from either α‐ or β‐secretase cleavage. In fact, the N‐terminal cysteine‐rich region of APP (residues 18–194) was as effective as the entire sAPP region in the inhibition of platelet activation. The inhibitory activity of sAPP correlated with a significant reduction in the agonist‐induced production of the arachidonic acid (AA) metabolites thromboxane B2 and prostaglandin E2. However, sAPP did not affect AA‐induced platelet aggregation or secretion, indicating the enzymatic conversion of AA was not inhibited. The addition of a threshold dose of AA reversed the sAPP‐inhibition of agonist‐induced platelet activation. This suggests that sAPP decreases the availability of free AA, although the mechanism is not yet known. These data provide evidence that the release of sAPP upon platelet degranulation may result in negative feedback regulation during platelet activation.
Human platelet antigen genotyping using a fluorescent SSCP technique with an automatic sequencer
Authors: Quintanar AndrÉ , Jallu Vincent , Legros Yann , Kaplan CÉcile
Published: March 2003

The typing of human platelet antigens (HPA) can be useful in many clinical situations such as neonatal alloimmune thrombocytopenia, post‐transfusion purpura, and platelet transfusion refractoriness. The fluorescent‐based single‐strand conformation polymorphism (F‐SSCP) technique is a fast and convenient way to perform HPA genotyping. Universal sequences from phage M13 were introduced at both ends of specific PCR‐products by using 5′‐tailed primers. A short second round of PCR with universal primers coupled to Cy‐5 enabled the PCR‐products to be fluorescently labelled. F‐SSCP was performed by gel electrophoresis on an automated fluorescent DNA analyser. Genotyping of the three major HPA systems carried by the GP IIb–IIIa complex showed the F‐SSCP technique to be accurate and reliable. A single gel procedure has been sufficient to detect HPA genetic polymorphisms tested to date. Neither restriction enzyme, radioactive material, nor any other hazardous chemicals such as ethidium bromide were required. This technique enabled us to genotype HPA‐1, ‐3 and ‐4 alleles easily and to diagnose materno‐fetal incompatibility in a rare alloantigenic system. F‐SSCP is a promising technique for the detection of new mutations and/or DNA polymorphisms on a large scale.
In‐vivo immune responses to idiotypic VH complementarity‐determining region 3 peptide vaccination in B‐cell non‐Hodgkin's lymphoma
Authors: Wen Yue‐Jin , Lim Seah H.
Published: March 2003

Idiotypic IgM expressed by B‐cell non‐Hodgkin's lymphoma (NHL) is clone specific and an ideal tumour antigen for immune targeting. We previously showed that repeated rounds of in vitro stimulation of peripheral blood mononuclear cells (PBMC) with autologous idiotypic heavy variable (VH) complementarity‐determining region (CDR)‐3 peptide produced T‐cell lines able to proliferate, produce Th1 cytokines and kill autologous target cells pulsed with the CDR3 peptide. Furthermore, fresh autologous lymphoma cells were lysed by these T‐cell lines, suggesting a potential for the clinical use of the CDR3 peptide for lymphoma vaccination. In this study, we determined the specific immune responses of a patient following vaccination with VH CDR3 peptide to determine if similar immune responses observed in vitro could be elicited in vivo. The patient received three fortnightly subcutaneous injections of idiotypic CDR3 peptide. The injection sites developed inflammation after the second and third vaccinations. Peripheral blood T cells were able to proliferate and produce IFN‐γ upon rechallenge with the CDR3 peptide in vitro. Cytotoxic T lymphocytes (CTLs) for autologous CD3‐depleted CDR3‐pulsed PBMC were detected, but only after the first vaccination. T‐cell lines generated after vaccination could lyse peptide‐pulsed autologous target cells. We also detected the production of anti‐CDR3 peptide antibodies in the patient's serum. We conclude that naked CDR3 peptide vaccination can result in the generation of potentially beneficial specific immune responses as predicted by in vitro studies.
Ultrastructural characteristics and lysozyme content in hypergranular and variant type of acute promyelocytic leukaemia
Authors: Shaft Dina , Shtalrid Mordechai , Berebi Alain , Catovsky Daniel , Resnitzky Peretz
Published: March 2003

We investigated the electronmicroscopic (EM) features and cellular lysozyme (LZ) content in 16 cases of acute promyelocytic leukaemia (APL): 11 cases of the hypergranular form (M3) and five cases of the microgranular variant (M3‐V). The main EM features in all cases were: irregular, folded or bilobed nuclei, many cytoplasmic granules, distended rough endoplasmic reticulum (RER) cisternae which, in some cases, presented as stellate forms (more frequent in M3‐V), and bundles of cytoplasmic microfilaments. Many Auer rods were present in M3 cases and few in M3‐V; most of these disclosed parallel tubular arrays (PTA) with a varied periodicity ranging from 13 to 26 nm. There was a significant difference between M3 and M3‐V (P<0.0001) in both the number of granules per cell section (62.9 ± 34.5 v 38.0 ± 23.6) and in the granule section area (0.044±0.033 v 0.026±0.015 μm2). In some cases, mainly in M3‐V, we found cells with large granules containing PTA which probably represent poorly developed Auer rods. Intracellular LZ content assayed by a post‐embedding immunogold method, showed high granular LZ density (in the range of that found in M4 and M5) in M3 cells and very low granular LZ content in M3‐V. This study adds new objective parameters for the diagnosis of these two types of APL and provides new information on their LZ pattern of expression.
Arsenic trioxide inhibits growth of human T‐cell leukaemia virus type I infected T‐cell lines more effectively than retinoic acids
Authors: Ishitsuka Kenji , Hanada Shuichi , Suzuki Shinsuke , Utsunomiya Atae , Chyuman Yoshiko , Takeuchi Syogo , Takeshita Taketsugu , Shimotakahara Sigemi , Uozumi Kimiharu , Makino Torahiko , Arima Terukatsu
Published: March 2003

Adult T‐cell leukaemia (ATL) is difficult to cure using conventional therapies. Recently the therapeutic possibility of retinoic acids (RA) has been reported. In this study, suppression of in vitro growth of human T‐cell leukaemia virus type I (HTLV‐I) infected T‐cell lines and fresh ATL cells by arsenic trioxide (As2O3) were evaluated by comparison with a series of RA derivatives. Proliferation of four HTLV‐I‐infected T‐cell lines was significantly reduced within 72 h by 1.0 μmol/l As2O3. Growth of two out of four HTLV‐I‐infected T‐cell lines was also inhibited by 1.0 μmol/l RA, but to a lesser extent than by As2O3. The mechanism of this growth inhibition was due to the induction of apoptosis. Apoptosis was also induced in fresh ATL cells from patients by As2O3, but far less by RA. As described in patients with acute promyelocytic leukaemia, 1.0 μmol/l of As2O3 can be safely achieved in the serum of patients; however, it is difficult to maintain this concentration of RA. In conclusion, As2O3 has therapeutic potential for the treatment of ATL and may be far more clinically beneficial than RA.
Systemic mastocytosis associated with acute myeloid leukaemia: report of two cases and detection of the c‐kit mutation Asp‐816 to Val
Authors: Sperr  , Walchshofer  , Horny  , Födinger  , Simonitsch  , Fritsche‐Polanz  , Schwarzinger  , Tschachler  , Sillaber  , Hagen  , Geissler  , Chott  , Lechner  , Valent
Published: March 2003

A subset of patients with systemic mastocytosis (SM) develop acute myeloid leukaemia (AML). However, little is known about the biology of such leukaemias and their relationship to the mast cell (MC) lineage. We report on two female patients who suffered from SM and AML. According to FAB criteria, the leukaemias were classified as AML‐M4 (patient 1) and AML‐M0 (patient 2). The coexistence of the two distinct neoplasms (AML and SM) was demonstrable by immunostaining of serial bone marrow (BM) sections with monoclonal antibodies (mAb). In particular, the MC infiltrates were found to react with mAb against MC‐tryptase and MC growth factor receptor c‐kit (CD117), but not with mAb to CD15 or CD34. In contrast, the AML blasts were immunoreactive for CD15 (patient 1) or CD34 (patient 2), but did not express tryptase. The c‐kit point mutation Asp → Val at codon 816, considered to play a role in the transformation of MC progenitors, was detected in patient 1 in a BM cell fraction containing 4% MC. However, no c‐kit mutation was found in pure AML blasts (<1% MC). These findings argue against an evolution of the AML clone from neoplastic MC or MC‐committed progenitors.
Clinical utility of immunoglobulin heavy chain gene rearrangement identification for tumour cell detection in multiple myeloma
Authors: Swedin Agneta , Lenhoff Stig , Olofsson Tor , Thuresson Britt , Westin Jan
Published: March 2003

In an attempt to define the clinical utility of immunoglobulin heavy chain (IgH) gene rearrangement identification for tumour cell detection in multiple myeloma, we investigated 36 consecutive newly diagnosed patients intended for high‐dose chemotherapy in a study protocol. After identification of the IgH rearrangement, an allele specific oligonucleotide (ASO) was constructed and used in a semiquantative PCR for minimal residual disease (MRD) evaluation. The myeloma‐specific IgH gene rearrangement could be identified and an ASO primer constructed in 24 (67%) of the patients. All of these patients underwent transplantation; 22 were autologous, of whom three had PCR‐negative stem cell harvests, and two were allogeneic. 10 patients achieved a clinical complete response (CR) and five were PCR negative in sequential bone marrow analyses. In patients not achieving CR, PCR negativity was occasionally found, but in general the PCR results reflected the clinical status of the patients. No consistent relationship between the bone marrow MRD status and the clinical course was found, and early relapses occurred also in PCR‐negative patients.
Regulation of human erythropoietin gene induction by upstream flanking sequences in transgenic mice
Authors: Köchling  , Curtin  , Madan
Published: March 2003

Human erythropoietin (Epo) gene expression is inducible by hypoxia or anaemia in the kidney and liver. Previous transgenic mouse experiments have demonstrated that sequences required for Epo gene induction in the kidney reside in a 7.8 kb Bam HI fragment located 6 kb upstream of the gene. To sublocalize these sequences, we performed Desoxyribonuclease I (DNAse I) mapping studies using transgenic mice which carried this DNA fragment. These studies revealed a DNAse I hypersensitive site (DNAse I HS) located 4.6 kb from the upstream end of the 7.8 kb fragment in anaemic kidney and liver samples. Sequence analysis of the region encompassing the DNAse I HS revealed an element with remarkable homology to the 3′ Epo gene hypoxia‐inducible enhancer. This suggested the presence of an additional regulatory element that contributes to the control of hypoxia‐inducible Epo gene expression in kidney and liver. We constructed transgenic mice containing the human Epo gene linked to either the 5 kb upstream or 2.5 kb downstream portion of the 7.8 kb fragment. Inducible expression was limited to the liver. Thus, neither fragment was alone sufficient to confer kidney inducible expression. These findings indicate that sequences more than 8.5 kb upstream of the Epo gene are required for kidney‐specific induction. They suggest that either those sequences reside in an 0.3 kb Hind III fragment located between the 5 kb and the 2.5 kb fragments or that sequences in the 5 kb or 0.3 kb fragments must interact with sequences in the 2.5 kb fragment to allow Epo gene induction in the kidney.
Mobilization of primitive haemopoietic progenitor cells and stem cells with long‐term repopulating ability into peripheral blood in mice by pegylated recombinant human megakaryocyte growth and development factor
Authors: Torii Yoshifumi , Nitta Yuko , Akahori Hiromichi , Tawara Tomonori , Kuwaki Tomoaki , Ogami Kinya , Kato Takashi , Miyazaki Hiroshi
Published: March 2003

We investigated in detail the effect of pegylated recombinant human megakaryocyte growth and development factor (PEG‐rHuMGDF) on peripheral blood progenitor cell (PBPC) mobilization in male BDF1 mice. Treatment with PEG‐rHuMGDF for 5 d stimulated a striking expansion of the circulating levels of multiple types of colony‐forming units in culture (CFU‐c), including CFU‐granulocyte‐macrophage, CFU‐megakaryocyte, burst‐forming units‐erythroid, and multipotent CFU‐c, and primitive day‐12 CFU‐spleen. All of these progenitors were mobilized into the peripheral blood (PB) with similar kinetics; their numbers peaked after the cessation of treatment and then declined earlier than platelet numbers peaked. The maximal increase in any of the four CFU‐c in the PB was attained with at least 300 μg/kg/d of PEG‐rHuMGDF, whereas peripheral platelet counts plateaued at 30 μg/kg/d. Adoptive transfer with PB from PEG‐rHuMGDF‐treated donor mice resulted in greater survival of lethally irradiated recipients. The majority of the recipients that survived at 187 d after transplantation with PEG‐rHuMGDF‐mobilized PB showed significant donor engraftment at the progenitor cell level. The combined administration of appropriate doses of PEG‐rHuMGDF and recombinant human granulocyte colony‐stimulating factor induced a synergistic increase in the circulating levels of the four CFU‐c compared to either factor alone. These results indicate that PEG‐rHuMGDF as a single agent can mobilize a full spectrum of PBPCs in mice.
HIV‐1 gp120 induces the activation of both c‐fos and c‐jun immediate‐early genes in HEL megakaryocytic cells
Authors: Gibellini Davide , Re Maria Carla , Bassini Alessandra , Guidotti Lia , Catani Lucia , La Placa Michele , Zauli Giorgio
Published: March 2003

We have previously demonstrated that the addition in culture of recombinant HIV‐1 IIIB envelope gp120 affects the survival/growth of pluripotent haemopoietic progenitors, and, in particular, of those committed towards the megakaryocytic lineage. To characterize some of the molecular mechanisms involved in this phenomenon, we investigated the expression of members of the activating protein‐1 (AP‐1) complex in the HEL megakaryoblastic cell line. Following the treatment of HEL cells with recombinant IIIB envelope gp120, we noticed: (i) increased levels of endogenous c‐fos and c‐jun mRNA and proteins, (ii) activation of both c‐fos and c‐jun promoters, and (iii) a very rapid stimulation of a MAPK/ERK pathway.
Identification of a novel mutation in a non‐Jewish factor XI deficient kindred
Authors: Alhaq Anwar , Mitchell Michael , Sethi Meera , Rahman Salman , Flynn Geraldine , Boulton Peter , Caeno Gianfranco , Smith Mark , Savidge Geoffrey
Published: March 2003

The role of factor XI (FXI) in blood coagulation has been clarified in recent years by descriptions of FXI‐ deficient patients who are prone to excessive bleeding after haemostatic challenge. We have studied a large kindred of an Italian FXI‐deficient patient with a previously undescribed mutation. The propositus, a 68‐year‐old woman, presented with a cerebral thromboembolic event but had no history of bleeding (FXI activity 1.6 U/dl). A sensitive ELISA failed to detect FXI antigen in the propositus. Sequence analysis of the entire FXI gene revealed a TGG to TGC transversion in codon 228 of exon 7 (FXI‐W228C). This missense mutation results in a Trp to Cys substitution within the third apple domain of FXI. We conclude that this novel mutation occurred in a structurally conserved region and may therefore have interfered with either chain folding and secretion or stability of FXI and was responsible for the inherited abnormality seen in this kindred. It is unclear why this kindred does not exhibit a bleeding tendency but it may correlate with a FXI‐like antigen and factor IX binding activity expressed on platelets.
Day‐care management of sickle cell painful crisis in Jamaica: a model applicable elsewhere?
Authors: Ware M. A. , Hambleton I. , Ochaya I. , Serjeant G. R.
Published: March 2003

In the U.K. and the U.S.A., painful crises account for 80–90% of sickle‐cell‐related hospital admissions, with average durations of 5–11 d. In Jamaica, many severe painful crises are managed in a day‐care centre. Patients (n=1160) with homozygous sickle cell (SS) disease aged 18 years and over were registered with the clinic during a 1‐year study period. Of these, 216 patients with 476 painful crises attended the day‐care facility for a total of 686 d. Most patients (119 or 55.1%) had single crises and for most crises (338 or 71%), patients attended for only 1 d, when they were given bed rest, assurance, rehydration and analgesia. Patients with complicated painful crises were usually referred for admission after initial pain relief and the rest were monitored during the day. In the evening they were given the option of hospital admission or allowed home with oral analgesia. Hospital admission for complicated painful crises or inadequate pain relief occurred in 42 (8.8%) crises and home management in 434 (91.2%) crises. Of 186 patients initially selecting home management, 20% returned for further day‐care and five (2.7%) died during subsequent admission for that painful crisis, one without other known complications, two with acute chest syndrome (one associated with Salmonella septicaemia), another with Salmonella septicaemia, and one with dengue haemorrhagic fever. With suitable oral analgesia, adequate education and support, the majority of severe painful crises in SS disease in Jamaica have been managed on an outpatient basis. This model of patient care may merit assessment in other communities where painful crises are a common clinical problem.
Complement sensitivity of erythrocytes in a patient with inherited complete deficiency of CD59 or with the Inab phenotype
Authors: Shichishima Tsutomu , Saitoh Yurie , Terasawa Takashi , Noji Hideyoshi , Kai Tatsuyuki , Maruyama Yukio
Published: March 2003

We investigated the complement sensitivity of erythrocytes from three patients, one with inherited complete deficiency of CD59, one with the Inab phenotype, and one with paroxysmal nocturnal haemoglobinuria (PNH). The complement lysis sensitivity units on the erythrocytes were 11.7, 4.6, and 47.6 for inherited CD59 deficiency, Inab phenotype, and PNH, respectively. Two‐colour flow cytometric analysis showed that the erythrocytes from the three patients consisted of a single population negative for CD59, negative for decay accelerating factor (DAF), and negative for both proteins, respectively. In addition, only the Inab phenotype patient had no haemolysis in vivo. These facts suggest that CD59 deficiency plays a more important role than DAF deficiency in complement‐mediated haemolysis in vitro and in vivo, and that deficiency of both proteins, but not CD59 or DAF alone, causes complement sensitivity corresponding to that of PNH III erythrocytes in vitro.
Magnetic resonance imaging in myelofibrosis and essential thrombocythaemia: contribution to differential diagnosis
Authors: ROZMAN CIRIL , CERVANTES FRANCISCO , ROZMAN MARÍA , MERCADER JOSEP‐MARÍA , MONTSERRAT EMILIO
Published: March 2003

To ascertain the value of magnetic resonance (MR) imaging in the differential diagnosis between myelofibrosis (MF) and essential thrombocythaemia (ET), 38 patients were analysed. 20 patients had MF (idiopathic myelofibrosis, 15 cases; post‐ET myelofibrosis, four cases; post‐polycythaemic MF, one case) and 18 ET. Mean age was 61.5 years (range 30–89) for patients with MF and 60.9 years (range 26–83) for ET patients. MR imaging was performed in the dorsal vertebrae in all cases, and also in both femurs in 25 of the patients. In most ET cases the MR signal of the dorsal vertebrae was not modified, whereas it was markedly reduced in MF (P = 0.0000001). With regard to femoral marrow, it was usually fatty in ET, with an absent to moderate degree of reconversion seen in the 14 cases analysed, contrasting with the marked degree of reconversion noted in 10/11 patients with MF (P = 0.000007). An inverse correlation was demonstrated between the vertebral signal and the degree of femoral reconversion. These differences were due to the fact that in ET the bone marrow adipose tissue is grossly preserved, whereas in MF it is usually markedly decreased or absent.

The above results indicate that MR imaging is a useful tool for the differential diagnosis of ET and MF, with the usefulness of this technique increasing when vertebral and femoral bone marrow studies are combined.
Intermediate‐dose intravenous melphalan and blood stem cells mobilized with sequential GM+G‐CSF or G‐CSF alone to treat AL (amyloid light chain) amyloidosis
Authors: COMENZO RAYMOND L. , SANCHORAWALA VAISHALI , FISHER CATHERINE , AKPEK GORGUN , FARHAT MAHA , CERDA SANDRA , BERK JOHN L. , DEMBER LAURA M. , FALK RODNEY , FINN KATHERINE , SKINNER MARTHA , VOSBURGH EVAN
Published: March 2003

AL amyloidosis patients ineligible for dose‐intensive melphalan (200 mg/m2) were enrolled on a phase II trial to be treated with two cycles of intermediate‐dose melphalan (IDM 100 mg/m2) and mobilized blood stem cells (BSC). For mobilization patients were randomized to either GM‐CSF 250 μg/m2 for 3 d followed by G‐CSF 10 μg/kg for 3 d (GM+G), or G‐CSF 10 μg/kg for 6 d (G‐alone), with leukaphereses on days 5, 6 and 7. To minimize morbidity, we planned to support each cycle with 3.5 × 106 CD34+ cells/kg and had a collection target of 7 × 106 CD34+ cells/kg. Those who did not achieve the target were treated with one cycle of IDM. 30 patients, a median of 62 years old and 7 months from diagnosis, were enrolled. Both mobilization regimens were generally well tolerated, and similar in terms of CD34+ cells and CFU‐GM collected, but only 6/28 patients achieved the collection target (GM+G four, G‐alone two). Despite a 19% incidence of grade 4 toxicities, IDM therapy was well tolerated. At a median follow‐up of 24 months (19–36) 57% of patients had survived, 17% with durable complete haematological responses and 40% with improved or stable amyloid organ involvement, including 3/9 patients with predominant cardiac amyloid who are alive 2–3 years after treatment. The 100 d mortality was 20%. In conclusion, no definitive differences were identified between the mobilization regimens and IDM was an active regimen in AL for selected patients.
Leukaemic presentation of small cell variant anaplastic large cell lymphoma: report of four cases
Authors: Bayle Chantal , Charpentier Agnès , Duchayne Eliane , Manel Anne‐Marie , Pages Marie‐Pierre , Robert Alain , Lamant Laurence , Dastugue Nicole , Bertrand Yves , Dijoud Frédérique , Emile Jean‐François , Machover David , Brugières Laurence , Delsol Georges
Published: March 2003

We report four cases of a rare subtype of CD30‐positive anaplastic large cell lymphoma (ALCL) with a predominant small cell component (small cell variant of ALCL) presenting with a leukaemic feature. Lymph node biopsy showed malignant cells of varying size with a predominant population of small to medium‐sized malignant cells associated with large anaplastic cells strongly positive for CD30 and epithelial membrane antigen (EMA). Both large and small cells were reactive with antibody ALK1, which recognizes the chimaeric NPM‐ALK protein associated with the t(2;5)(p23;q35). All patients presented with hyperleucocytosis with atypical small lymphocytes. Bone marrow involvement was detected on both aspirate and bone marrow trephine where scattered malignant cells were only demonstrated by immunostaining for CD30 and ALK protein. Atypical cells in peripheral blood, lymph node and skin biopsies showed a T or null cell phenotype. Cytogenetic analysis of blood, bone marrow and/or lymph node revealed the t(2;5)(p23;q35) characteristic of ALCL. The patients responded to chemotherapy but showed early relapse without abnormal cells in peripheral blood.

This report shows that the small cell variant of ALCL may have a leukaemic presentation with peripheral blood involvement by atypical lymphocytes and provides evidence that, in the small cell variant of ALCL, the small cell component is a part of the malignant clone.
Pulmonary Aspergillus colonization in humans and its impact on management of critically ill patients
Authors: Lass‐Flörl Cornelia , Salzer Georg M. , Schmid Thomas , Rabl Walter , Ulmer Hanno , Dierichi Manfred P.
Published: March 2003

Samples of lung tissues were obtained and analysed for Aspergillus carriage in 56 patients undergoing thoracic surgical intervention and 18 people who had an unexpected death. Out of 74 samples, 46 (63%) had evidence of pulmonary fungal colonization. The surgery population had a rate of 62% of fungal growth, Aspergillus was present in 39%. The autopsy population had a rate of 61% of fungal colonization, Aspergillus was present in 41%. In these cases eradication of fungal spores residing in the lung prior to aggressive chemotherapy and prevention of further spore uptake during hospitalization is indispensable in preventing pulmonary aspergillosis.
Synthesis of Rh Fv phage‐antibodies using VH and VL germline genes
Authors: Hughes‐Jones Nevin , Bye Jacqueline , Gorick Barbara , Marks James , Ouwehand Willem
Published: March 2003

Antibodies to the D antigen of the Rh system use a restricted set of immunoglobulin V and J gene segments, especially VH DP50 and DP63, JH6, Vλ DPL16 and Jλ 2/3. These gene segments may confer a natural affinity on the antibodies for the D antigen and this hypothesis has been tested by constructing two single‐chain Fv phage‐antibody libraries based on the germline gene segments DP50 and DP63; structural variability was obtained by insertion of 11 amino acids in random sequence in the VHCDR3. 10 anti‐D antibodies were selected from these libraries, each with a unique VHCDR3. In contrast, selections with the CcEe antigens produced antibodies reacting with the Rh polypeptide molecules but without strict blood group specificity. One of these latter DP50‐based antibodies was converted into 12 different antibodies with specificity for E by replacing the original germline light chain with chains from a rearranged L chain library. The CDR1 and CDR2 sequences of the DP50‐based antibodies were common to both anti‐D and anti‐E molecules; differentiation between D and E specificity was dependent on VHCDR3 sequences and their correct pairing with an appropriate L chain.
Purine analogues kill resting lymphocytes by p53‐dependent and ‐independent mechanisms
Authors: Pettitt A. R. , Clarke A. R. , Cawley J. C. , Griffiths S. D.
Published: March 2003

To resolve the controversy concerning the role of p53 in the killing of resting lymphocytes by purine nucleoside analogues, we examined the cytotoxic effects of chlorodeoxyadenosine, fludarabine and deoxycoformycin (plus deoxyadenosine) on unstimulated spleen cells from p53‐knockout versus wild‐type mice.

p53‐knockout cells were more resistant to all three nucleosides than were wild‐type cells. However, substantial killing still occurred in the absence of p53, indicating that purine analogues can kill resting lymphocytes by both p53‐dependent and ‐independent mechanisms.

We suggest that these results are relevant to chronic lymphoid malignancies, and that characterization of the p53‐independent component of nucleoside action may indicate potential ways of overcoming therapeutic resistance.
Monocyte dysfunction in patients with multiple myeloma and lymphoplasmacytic disorders is related to serum paraprotein levels
Authors: Mainwaring C. Jason , Williams Marc A. , Singer Charles R. J. , LUSH Richard J. , Smith J. Graham , Haynes Clive L. , Kelsey Stephen M.
Published: March 2003

We have investigated monocyte function in 30 patients with lymphoplasmacytic disorders and in 21 age and sex matched normal controls. Marked abnormalities of all facets of monocyte function were demonstrated in six patients with multiple myeloma (MM) and a single patient with Waldenström's macroglobulinaemia (WM) plus significant paraproteinaemia. Serious infection occurred in three of these patients. An inverse relationship between the level of the serum paraprotein and impairment of monocyte phagocytosis plus killing of Candida albicans was observed. Crossover studies suggested that these abnormal findings were constitutive and not reversed by removal of the serum paraprotein. The data suggest that monocyte function is constitutively abnormal in patients with MM and can be further, but reversibly, inhibited by high paraprotein levels. Further research is required to confirm these findings, ascertain whether monocyte function can be normalized using chemotherapy or growth factors, and if so, whether their tumouricidal functions could be harnessed in the treatment of this currently incurable condition.
Myeloid differentiation of human cord blood CD34+ cells during ex vivo expansion using thrombopoietin, flt3‐ligand and/or granulocyte‐colony stimulating factor
Authors: Yoo Eun‐Seon , Ryu Kyung‐Ha , Park Hae‐Young , Seong Chu‐Myung , Chung Wha‐Soon , Kim Seung‐Cheol , Choi Yong‐Mook , Hahn Myong‐Joon , Woo So‐Youn , Seoh Ju‐Young
Published: March 2003

We investigated the phenotypic changes of human umbilical cord blood (CB) CD34+ cells during ex vivo expansion using thrombopoietin (TPO), flt3‐ligand (FL), and/or granulocyte‐colony stimulating factor (G‐CSF). During ex vivo expansion of CD34+ cells isolated from human CB for up to 5 weeks, surface expression of molecules on the cultured cells including CD64 (FcγRI), CD32 (FcγRII), CD16 (FcγRIII), CD11b (MAC‐1) and CD18 (β2‐integrin) was analysed by flow cytometry along with simultaneous measurement of apoptosis by 7‐aminoactinomycin D staining method. CD64, CD32 and/or CD18 expressing cells appeared in the cultures both with and without the addition of G‐CSF until the tenth day. However, without G‐CSF, CD16+ fractions did not appear and CD11b+ fractions were not maintained. With G‐CSF, the CD16+ or CD11b+ fractions appeared only from the second week. These results suggest that G‐CSF is necessary for the late stage of myeloid maturation during which CD16 and CD11b are expressed.
A novel mutation of the erythroid‐specific δ‐aminolaevulinate synthase gene in a patient with X‐linked sideroblastic anaemia
Authors: Harigae Hideo , Furuyama Kazumichi , Kimura Akiro , Neriishi Kazuo , Tahara Nobuyasu , Kondo Masao , Hayashi Norio , Yamamoto Masayuki , Sassa Shigeru , Sasaki Takeshi
Published: March 2003

A novel missense mutation, A1754G, in exon 11 of the erythroid‐specific δ‐aminolaevulinate synthase gene (ALAS2) was identified in a Japanese male with sideroblastic anaemia. ALAS activity in bone marrow cells of the patient was reduced to 53.3% of the normal control. Consistent with this finding, activity of a bacterially expressed ALAS2 mutant protein harbouring this mutation was 19.5% compared with the normal control, but was increased up to 31.6% by the addition of pyridoxal 5′‐phosphate (PLP) in vitro. RFLP analysis with Bsp HI restriction revealed that his mother was a carrier of the mutation. These findings suggest that A1754G mutation was inherited in this family in a manner consistent with X‐linked inheritance, and is responsible for sideroblastic anaemia in the patient.
In vitro evaluation of the haemopoietic defect of CD34+ cells from patients with acute myeloid leukaemia in first remission
Authors: Herman  , Van Bockstaele  , Ferrant  , Straetmans
Published: March 2003

Haemopoietic cells from patients with acute myeloid leukaemia (AML) in first complete remission (CR1) show in vitro a haemopoietic defect and a decreased expansion potential. To better characterize this haemopoietic defect, CR1 AML and normal CD34+ cells were analysed for immunophenotype, viability, cell cycle and progenitor content before and during expansion culture in stroma‐conditioned medium supplemented with cytokines. The production of haemopoiesis inhibitor by patient cells and the influence of high concentrations of stem cell factor (SCF) and Flt3‐ligand (FL) on cell survival and ex vivo expansion potential were also studied. Before expansion, patient CD34+ cells showed viability and cell‐cycle phase distribution similar to normal but lower percentages of CD34+DR− or CD34+CD38− cells and lower progenitor content. After 48 h of culture ±30% of patient cells had died regardless of the cytokine combination used, whereas only 15% of normal cells died. After 7 d of culture, viability and cell cycle analyses showed comparable data for normal and patient samples. Co‐culture of patient and normal cells did not show any evidence for haemopoiesis inhibitor production by patient cells. Even at high cytokine concentrations, a low progenitor expansion and a decrease in CD34+ cell numbers was observed for patient samples in contrast to normal samples. In conclusion, CR1 AML CD34+ cells showed excessive early cell mortality. No evidence for cell‐cycle arrest or haemopoiesis inhibitor production was shown. SCF and FL used at high concentrations did not correct the patient cell expansion defect.
Immunologic response to recombinant hirudin in HIT type II patients during long‐term treatment
Authors: Huhle  , Hoffmann  , Song  , Wang  , Heene  , Harenberg
Published: March 2003

We prospectively investigated 27 patients with heparin‐induced thrombocytopenia (HIT) type II who were subsequently treated with r‐hirudin. Patients with venous or arterial thromboembolism were treated with activated partial thromboplastin time (aPTT)‐controlled intravenous r‐hirudin (n = 19; mean 19.3 d) followed by subcutaneous r‐hirudin (n = 6; mean 22.5 d) and oral anticoagulation. Patients without thromboembolism were treated with subcutaneous r‐hirudin (n = 8; mean 25.9 d). Four patients were readmitted to subcutaneous r‐hirudin for a mean duration of 32 d. The incidence of r‐hirudin antibodies was 84% for intravenously treated patients and 50% in subcutaneously treated patients. The patients (n = 27) showed a 74% overall incidence of r‐hirudin antibodies, mainly of the IgG‐subclass, without seroconversion before day 6 and after day 32 of r‐hirudin treatment or during r‐hirudin treatment. None of the patients showed onset or recurrence of venous or arterial thromboembolism, systemic allergic reactions or IgE‐antibody development.

During intravenous and subcutaneous administration of r‐hirudin the aPTT and the ecarin clotting time was increased in the antibody‐positive patients compared to antibody‐negative patients. Therefore we assume that r‐hirudin antibodies may reduce r‐hirudin metabolism.
Long‐term follow‐up of patients with hairy cell leukaemia after treatment with pentostatin or cladribine
Authors: Dearden C. E. , Matutes E. , Hilditch B. L. , Swansbury G. J. , Catovsky D.
Published: March 2003

We report the long‐term follow‐up results on two groups of patients with hairy cell leukaemia (HCL) treated with either pentostatin (deoxycoformycin) or cladribine (2‐chlorodeoxyadenosine). 165 HCL patients received treatment with pentostatin (between 1986 and 1994), and 45 were treated with cladribine (between 1992 and 1997). Age and sex characteristics were similar in the two groups. 38 patients in the pentostatin group and 12 in the cladribine group were previously untreated. 22 patients in the cladribine group had received prior treatment with pentostatin; four were resistant, 17 had relapsed following partial (four) or complete (13) responses, and one was not evaluable for response. The response rates were the same in the two groups: 82% complete response (CR), 15% partial response (PR) for pentostatin and 84% CR, 16% PR for cladribine. Relapse rates were 24% for pentostatin and 29% for cladribine after median follow‐up of 71 and 45 months respectively. At 45 months, however, the relapse rate for pentostatin was only 9.7%. We found a statistically significant difference in the disease‐free interval (DFI) between the two groups suggesting that patients may relapse more quickly after cladribine. The majority of relapsed patients achieved second remissions following further therapy with either pentostatin or cladribine, with no evidence of cross resistance between the two agents. The 5‐year survival for all patients was 97% and treatment‐ related toxicity was low. We conclude that both pentostatin and cladribine induce durable remissions in the majority of HCL patients. Longer follow‐up is required to establish whether some patients are cured as there is no plateau in DFI, and which of these two agents may be the treatment of choice.