bluuebunny/crossref_metadata_2025 huggingface dataset:
In vivo haemoprotective activity of tetrapeptide AcSDKP combined with granulocyte‐colony stimulating factor following sublethal irradiation
Authors: Watanabe Tsutomu , Kelsey Linda S. , Yan Yun , Brown Gregory S. , Jackson John D. , Ewel Cynthia , Talmadge James E.
Published: November 2003

We report that acetyl‐n‐Ser‐Asp‐Lys‐Pro (AcSDKP), which removes progenitor cells from cell cycle, in combination with granulocyte‐colony stimulating factor (G‐CSF) can significantly improve myelorestoration following irradiation (7 Gy). Peripheral blood, spleen and bone marrow (BM) cell recovery and progenitor cell reconstitution [IL‐3‐responsive colony‐forming cells (CFC) and high proliferative potential colony‐forming cells (HPP‐CFC)] were studied. Studies on the optimal schedule of AcSDKP administration revealed maximal effects on progenitor cells when AcSDKP was administered as a continuous infusion for 3 d starting 24 h prior to irradiation and used in combination with G‐CSF. The numbers of CFC and HPP‐CFC in the BM were significantly increased following irradiation in mice receiving AcSDKP and G‐CSF as compared to either drug alone. The numbers of CFC in the spleen were significantly increased in mice receiving AcSDKP and G‐CSF on days 10 and 14 as compared to AcSDKP alone, but not G‐CSF. Similarly, CFC and HPP‐CFC in the spleen were significantly increased in mice receiving AcSDKP and G‐CSF on day 18 as compared to mice receiving PBS and G‐CSF. These studies suggest that AcSDKP in combination with G‐CSF may have potential for the protection of progenitor cells in patients undergoing intensive chemo‐ and/or radiotherapy.
In leukaemic CD5+ B cells the expression of BCL‐2 gene family is shifted toward protection from apoptosis
Authors: Gottardi D. , Alfarano A. , De Leo A. M. , Stacchini A. , Aragno M. , Rigo A. , Veneri D. , ZANOTTI R. , Pizzolo G. , Caligaris‐Cappio F.
Published: November 2003

This study further investigated the mechanisms that control apoptosis in leukaemic CD5+ B cells, and focused on the Bcl‐2 gene family. The pattern of expression of Bcl‐2, Bcl‐xL, Bcl‐xS and Bax genes, selected because of their interrelated role in the control of apoptosis, was analysed in a series of CD5+ B‐cell chronic lymphoid leukaemias.

Cells from 34 patients with chronic lymphoid leukaemia of B‐cell type (23 B‐chronic lymphocytic leukaemia (B‐CLL) and 11 mantle cell lymphoma (MCL) in leukaemic phase) were investigated. High levels of Bcl‐2 mRNA were observed by Northern blot and high levels of Bcl‐2 protein were detected by cytofluorograph analysis with a specific monoclonal antibody (MAb) in all cases. Strong Bax expression was detected by RT‐PCR in 20/23 B‐CLL cases; Bax was also observed in 8/11 MCL in leukaemic phase with variable degree of intensity. In both B‐CLL and MCL samples the presence of Bax protein was confirmed by cytofluorograph analysis. RT‐PCR detected high levels of Bcl‐xL in 16/23 B‐CLL and in 8/11 MCL in leukaemic phase, whereas Bcl‐xS was detectable in low to trace amounts respectively in 13/23 B‐CLL and in 6/11 MCL in leukaemic phase.

According to the functional role of Bcl‐2, Bcl‐xL, Bcl‐xS and Bax, these data indicate that the pattern of Bcl‐2 family genes expression in leukaemic CD5+ B cells is skewed toward prevention of apoptosis and may thus favour the relentless accumulation of CD5+ leukaemic B cells.
Epidemiological studies of spectrin mutations related to hereditary elliptocytosis and spectrin polymorphisms in Benin
Authors: Glele‐Kakai Clement , Garbarz Michel , Lecomte Marie‐Christine , Leborgne Sylvie , Galand Colette , Bournier Odile , Devaux Isabelle , Gautero Huguette , Zohoun Isidore , Gallagher Patrick G. , Forget Bernard G. , Dhermy Didier
Published: November 2003

We studied an African population in Benin and discovered an unexpectedly high frequency (1.6%) of hereditary elliptocytosis (HE) among the 1447 subjects studied. In approximately two‐thirds of HE individuals we identified molecular defects, primarily those in erythrocyte α‐spectrin (dupL154, L260P and L207P mutations), as well as a novel mutation of erythrocyte β‐spectrin (β‐W2061R mutation). We also identified the genetic basis of a previously identified protein polymorphism of the αIII domain of spectrin (R1331I mutation). The genetic background of HE in the African population was studied using a number of polymorphisms of the α‐spectrin gene, including the αIII domain polymorphism. These studies suggest that the HE mutations appear to have originated from separate genetic backgrounds in this population.
Changes in coagulation and fibrinolysis in childhood ALL: a two‐step dose reduction of one E. coli asparaginase preparation
Authors: Nowak‐Göttl Ulrike , Ahlke Elvira , Schulze‐Westhoff Petra , Boos Joachim
Published: November 2003

The influence of two different E. coli asparaginase (ASP) preparations on fibrinolytic proteins in childhood ALL was recently reported, demonstrating a clearly significant association between ASP activity and haemostatic changes. Since the Bayer preparation is no longer available for treatment of large series of patients with ALL, the present study was designed to prospectively evaluate coagulation and fibrinolytic changes in leukaemic children receiving different doses of Medac ASP, which is now available for treatment of childhood ALL. Leukaemic children in whom ASP Medac was administered at 3 d intervals in a two‐step dose reduction (5000 IU/m2, n = 10; 2500 IU/m2, n = 15) were compared with children who had received Bayer ASP 10 000 IU/m2 in the same time schedule in a former randomized trial; at the same venipuncture, blood samples for coagulation studies were obtained before each ASP administration together with serum samples for pharmacoinetic monitoring. Compared with Bayer ASP 10 000 IU/m2, patients receiving Medac ASP 5000 IU/m2 showed significantly decreased values of fibrinogen, plasminogen, and α2‐antiplasmin, along with significantly enhanced thrombin generation. Improvement occurred in children treated with 2500 IU/m2 Medac ASP; α2‐antiplasmin and D‐dimer no longer differed from the Bayer group. Since both patient groups showed complete asparagine depletion during the course of ASP administration, the lower dosage of 2500 IU/m2 administered at 3 d intervals should guarantee the specific metabolic therapy for ALL, leading to depletion of the circulating pool of asparagine.
Congenital dyserythropoiesis characterized by marked macrocytosis, vitamin B12‐ and folate‐independent megaloblastic change and absence of the defining features of congenital dyserythropoietic anaemia types I or III
Authors: WICKRAMASINGHE S. N. , ANDREWS V. E. , O'HEA A. M.
Published: November 2003

Four patients with congenital dyserythropoiesis characterized by marked macrocytosis, little or no anaemia, and vitamin B12‐ and folate‐independent megaloblastic erythropoiesis are reported. Their erythroblasts also showed various dysplastic changes but not those diagnostic for congenital dyserythropoietic anaemia (CDA) types I or III. The haematological features of the four patients, who included two siblings, resemble those of a previously reported patient and together these patients form a recognizable subgroup within those cases of CDA not belonging to CDA types I–III. In two of the cases studied, and possibly a third, the inheritance was as an autosomal recessive character.
Preceding standard therapy is the likely cause of MDS after autotransplants for multiple myeloma
Authors: Govindarajan Rangaswamy , Jagannath Sundar , Flick James T. , Vesole David H. , Sawyer Jeffrey , Barlogie Bart , Tricot Guido
Published: November 2003

Myelodysplastic syndromes (MDS) and acute myeloid leukaemia (AML) have been reported after autologous transplantation (AT) for lymphoma. It is not clear whether myeloablative therapy used in conjunction with autologous transplantation contributes to the development of MDS/AML or whether the conventional chemotherapy preceding the transplant, and administered over a prolonged period, causes these secondary malignancies. To address this issue, we examined 188 patients with multiple myeloma (MM) who had received AT. 71 patients with no more than one cycle of standard chemotherapy were enrolled in our Total Therapy program, designed to avoid exposure to alkylating agents prior to peripheral blood stem cell mobilization (group 1). The median duration of pre‐transplant therapy in group 1 was 7.6 months and significantly shorter than the 24 months of 117 patients (group 2) with more prolonged conventional therapy (P = 0.0001). All seven patients developing MDS post‐transplantation belonged to group 2 (P = 0.02); the median durations from initial therapy and first transplant were 66 months (range 38–86) and 24 months (range 9–39), respectively. Our findings provide evidence that prolonged standard‐dose alkylating agent therapy prior to transplantation, rather than autotransplant‐supported myeloablative treatment, is associated with development of MDS/AML. Stem cell damaging alkylator treatment should be avoided, not to compromise PBSC collection, but also to reduce the risk of treatment‐related MDS/AML.
A combined management protocol for patients with coagulation disorders infected with hepatitis C virus
Authors: Ahmed M. M. , Mutimer D. J. , Elias E. , Linin J. , Garrido M. , Hubscher S. , Jarvis L. , Simmonds P. , Wilde J. T.
Published: November 2003

The case notes of 394 adults with bleeding disorders registered at our centre together with those of the 72 patients who had died since 1971 were reviewed. 36/72 deceased patients had evidence of HCV infection. Liver decompensation was present at time of death in six. 274 (70%) of the currently registered patients had received factor concentrate or cryoprecipitate and 174 of these were screened for HCV infection. 76% of tested patients were RIBA positive. 87% of RIBA‐positive patients were RT‐PCR positive. 50 RIBA‐positive patients, including nine who were HIV infected, have undergone percutaneous liver biopsy following appropriate factor infusion with no complication. The biopsy was assessed using a Histological Activity Index (HAI) ranging from 0 to 13. Patients with HAI ≥6 were offered treatment with interferon. Patients with HAI<6 were followed up with a view to re‐biopsy in 2–3 years to assess progression. The median HAI was 4.5 (range 0–10) with HAI ≥6 in 13 cases (27%). HAI was not correlated with duration of infection, haemophilia severity, RT‐PCR status, HIV status or HCV genotype. Liver biopsy, a safe procedure in our hands, is an important investigation in HCV‐infected patients to assess suitability for interferon therapy.
Adriamycin stimulates proliferation of human lymphoblastic leukaemic cells via a mechanism of hydrogen peroxide (H2O2) production
Authors: Yang Min , Nazhat Najdat B. , Jiang Xurong , Kelsey Stephen M. , Blake David R. , Newland Adrian C. , Morris Christopher J.
Published: November 2003

It is becoming clear that adriamycin cytotoxicity may be mediated by semiquinone‐free radicals derived from the drug itself and reactive oxygen species (ROS). Recent evidence supports the concept that low concentrations of ROS are able to stimulate cell proliferation, and, based on the observation that subtoxic concentrations of adriamycin can also induce cell proliferation, we hypothesize that low concentrations of adriamycin stimulate cell proliferation by a ROS generation mechanism. We have employed spin‐trapping and electron spin resonance (ESR) spectroscopy to investigate the nature of the adriamycin‐generated ROS. The spin trap 3,5‐dibromo‐4‐nitrosobenzenesulphonate (DBNBS), which is oxidized in the presence of H2O2 and peroxidase enzymes, was used to produce a characteristic three‐line spectrum, and it was found that an identical spectrum was produced by human lymphoblastic leukaemic cells (CCRF‐CEM cells) after exposure to adriamycin. We tested our hypothesis further by exposing CCRF‐CEM cells to subtoxic concentrations of adriamycin (10−8, 10−9 and 10−10 M) and low concentrations of H2O2 (10−8, 10−9 and 10−10 M) and subsequently monitored cell proliferation. We found that low concentrations of both adriamycin and H2O2 significantly stimulate CCRF‐CEM cell proliferation. We therefore conclude that subtoxic concentrations of adriamycin are likely to induce cell proliferation via an H2O2 mediated mechanism.
Activated platelet aggregates in thrombotic thromboctyopenic purpura: decrease with plasma infusions and normalization in remission
Authors: Ahn Yeon S. , Jy Wenche , Kolodny Luciano , Horstman Lawrence L. , Mao Wei‐Wei , Valant Peter A. , Duncan Robert C.
Published: November 2003

Circulating activated platelet aggregates (aPA) were assayed by flow cytometry employing mAb α‐CD62p in eight patients with thrombotic thrombocytopenic purpura (TTP). Elevation of aPA was observed in all patients in active stages of TTP; aPA normalized in remission. Plasma infusions with plasmapheresis decreased aPA in responding patients. The rise and fall of aPA preceded relapses and improvements, respectively. These changes were seen prior to the traditional indicators, LDH, haematocrit, and platelet count. Incubation of plasma from TTP patients with normal whole blood induced formation of aPA; this effect was significantly greater than that of plasmas from ITP patient controls (P < 0.01), suggesting the presence of an aPA‐promoting factor in TTP plasma. Parallel experiments using a platelet aggregometer failed to detect effects of TTP plasma on normal blood. In summary, aPA appear to be a marker of disease activity, rising with relapse, falling with plasma therapy, and normalizing in remission. The flow cytometric assay of aPA is more sensitive than aggregometry in detecting the putative aPA‐promoting factor in TTP.
EBV‐positive non‐Hodgkin's lymphoma developing after phenytoin therapy
Authors: Garcia‐SuArez Julio , DOMINGUEZ‐FRANJO PURIFICACION , DEL CAMPO J. Francisco , HERRERO BEGOÑA , MUÑOZ M. ANGELES , PIRIS MIGUEL A. , PARDO ANA
Published: November 2003

We report the first case of Epstein Barr virus (EBV) positive non‐Hodgkin's lymphoma occurring 20 years after continuous phenytoin therapy for idiopathic epilepsy. This malignant lymphoproliferative disease was preceded by an phenytoin‐associated benign reactive lymphadenopathy. Serological findings suggested a chronic active EBV infection. Immunohistochemistry showed that neoplastic cells expressed B antigens (CD20, lambda) and the bcl‐2 protein. The presence of EBV‐encoded latent membrane protein 1 (LMP 1) was also expressed on neoplastic cells. Cytotoxic chemotherapy (CHOP) reversed the non‐Hodgkin's lymphoma.
Proliferation of precursor B‐lineage acute lymphoblastic leukaemia by activating the CD40 antigen
Authors: Planken E. V. , Dijkstra N. H. , Bakkus M. , Willemze R. , Kluin‐Nelemans J. C.
Published: November 2003

No reliable culture system exists for B‐lineage acute lymphoblastic leukaemia (ALL). Recently we found that many different mature B‐cell malignancies proliferate upon stimulation via the CD40 antigen, and this led us to investigate whether a similar CD40 activation on ALL cells could also induce proliferation. First, we measured CD40 expression in 21 ALL cases; all were CD40+, although mostly weak. Next, we triggered the CD40 antigen by anti‐CD40 antibodies and by a CD40 ligand‐expressing cell line. In addition, we measured the influence of IL‐3, IL‐4 and IL‐7 with and without these stimuli.  In 8/10 cases proliferation, measured by 3H‐thymidine incorporation, could be induced after CD40 crosslinking, especially in the presence of IL‐3. Stimulation via the CD40 ligand was more successful than using crosslinked anti‐CD40 antibodies. IL‐4 inhibited the spontaneous proliferation found in three cases, but stimulated proliferation after CD40 crosslinking. IL‐7 did not contribute to proliferation. Morphology, immunophenotyping and surface marker analysis, combined with DNA flow cytometry confirmed that the proliferation found could be ascribed to the ALL cells.  In conclusion, B‐lineage ALL cases are CD40+, and many can be cultured using CD40 stimulation and IL‐3.
Evaluation of ‘discordant maturation’ in chronic myeloid leukaemia using cultures of primitive progenitor cells and their production of clonogenic progeny (CFU‐GM)
Authors: Marley S. B. , Lewis J. L. , Scott M. A. , Goldman J. M. , Gordon M. Y.
Published: November 2003

The ‘discordant maturation hypothesis’ proposes that the most mature proliferating cells in chronic‐phase chronic myeloid leukaemia (CML) are responsible for the expansion of the Ph‐positive population. To evaluate this hypothesis we used a delta assay for primitive haemopoietic cells (PΔ assay for PΔ cells) which allows investigation of the kinetics of granulocyte‐macrophage progenitor (CFU‐GM) production. The frequencies of these primitive (PΔ) cells were similar in CML blood (14.5/105 mononuclear cells), CML marrow (17.3/105) and normal marrow (11.6/105) The average frequency in normal blood is only 0.58/106. The absolute numbers of PΔ cells in CML patients are therefore greatly increased. The average numbers of CFU‐GM produced by individual PΔ cells were reduced in CML blood (8.1) and marrow (11.6) compared with normal marrow (28.5). This is consistent with a reduced probability of differentiation at the single cell level in CML. Although the absolute number of CFU‐GM produced by individual CML PΔ cells was subnormal there was a relative increase in the number of day 7 CFU‐GM compared with the number of day 14 and 21 CFU‐GM, which agrees with the ‘discordant maturation hypothesis’. This bias towards day 7 colony formation could reflect accelerated maturation by the CFU‐GM produced by PΔ cells or, alternatively, the production of CFU‐GM with shorter than normal maturation pathways. Overall, these results suggest that discordant maturation does not by itself account for myeloid expansion in CML. It is more likely that myeloid expansion in CML is due mainly to an increase in the number of primitive haemopoietic progenitor cells.
Is apoptosis a massive process in myelodysplastic syndromes?
Authors: Lepelley Pascale , Campergue Laurence , Grardel Nathalie , Preudhomme Claude , Cosson Alain , Fenaux Pierre
Published: November 2003

We looked for increased apoptosis in fresh bone marrow aspirates in 40 cases of myelodysplastic syndrome (MDS), by detection of DNA fragmentation using TdT incorporation of nucleotides on 3′ ends of DNA (TUNEL technique). No DNA laddering was seen. In six cases (15%) the TUNEL technique showed a moderate increase in the percentage of apoptotic cells (2.5–5% in comparison with <2% in controls).

In seven of the 34 patients with normal findings by TUNEL analysis, apoptosis was reanalysed after short‐term (18 h) bone marrow culture without inducers of apoptosis. Increased apoptosis was shown in four of the seven cases by morphological analysis and/or the TUNEL technique. Increased apoptosis predominated on erythroblasts in three of them. The percentage of apoptotic cells, however, was <40% in all samples.

Our findings suggest that increased apoptosis can be detected in one half of MDS cases after cell culture. Furthermore, the precise relationship between increased apoptosis of myeloid precursors and cytopenias will have to be more precisely explored in MDS.
Recombinant factor IX secreted by transduced human keratinocytes is biologically active
Authors: Gerrard A. J. , Austen D. E. G. , Brownlee G. G.
Published: November 2003

We have been investigating the use of human keratinocytes as potential target cells for gene therapy for haemophilia B, with the aim of curing haemophilia by means of a factor IX secreting skin graft. Previous studies indicated that keratinocytes might be suitable cells, although a potential problem was that the recombinant factor IX secreted by transduced keratinocytes was found to be only 40% biologically active. We now report, using an alternative assay to test for activity, that the secreted factor IX appears to be almost fully active.
An IVS1–116 (A→G) acceptor splice site mutation in the α2 globin gene causing α+ thalassaemia in two Dutch families
Authors: Harteveld C. L. , Heister J. G. A. M. , Giordano P. C. , Batelaan D. , Delft P. v. , Haak H. L. , Wijermans P. W. , Losekoot M. , Bernini L. F.
Published: November 2003

We report the characterization of an α+‐thalassaemia determinant due to a transition A → G of the acceptor splice consensus site sequence (IVS1–116) of the first intron of the α2‐globin gene. The mutation, found in two apparently unrelated Dutch Caucasian families, was detected by DGGE analysis followed by direct sequencing. Haplotype analysis suggests a common origin of the mutation in both families. The disruption of the acceptor splice site consensus sequence interferes with the correct splicing and leads to the retention of the first intron in the abnormally spliced mRNA. The α+‐thalassaemia phenotype observed in the carriers is caused by the absence of functional mRNA which cannot be replaced by the abnormally spliced mRNA. The low amounts of abnormal mRNA found in reticulocytes is, most probably, due to the post‐transcriptional instability which follows the presence of a termination codon in the retained intronic sequence. This situation is often associated with a decreased mRNA stability as observed for several nonsense mutations of the β‐globin gene.
A pilot study of 220 mg/m2 melphalan followed by autologous stem cell transplantation in patients with advanced haematological malignancies: pharmacokinetics and toxicity
Authors: Moreau Philippe , Kergueris Marie‐France , Milpied NoE¨l , Le Tortorec Stéphane , Mahé Béatrice , Bulabois Claude‐Eric , Rapp Marie‐JosÉ , Larousse Claude , Bataille RÉgis , Harousseau Jean‐Luc
Published: November 2003

We studied the pharmacokinetics and toxicity of 220 mg/m2 melphalan (HDM 220) followed by autologous stem cell transplantation in 16 patients with advanced haematological malignancies. Pharmacokinetic parameters (mean values of steady‐state volume of distribution 14.6 l/m2, total body clearance 313 ml/min/m2, elimination half‐life 46 min) were the same as those of 140 or 200 mg/m2 melphalan in previous reports. HDM 220 was feasible. Extramedullary toxicity was mainly W.H.O. grade 4 mucositis (13/16 patients). The median duration of 41 d (10, not reached) of thrombocytopenia <25 × 109/l was long. In multiple myeloma the response rate was 89% in heavily pretreated patients, suggesting that HDM 220 could be considered earlier in the course of the disease as an alternative consolidation therapy.
Circulating thrombopoietin concentrations in thrombocytopenic patients, including cancer patients following chemotherapy, with or without peripheral blood progenitor cell transplantation
Authors: Meng Y. Gloria , Martin Thomas G. , Peterson Michael L. , Shuman Marc A. , Cohen Robert L. , Wong Wai Lee
Published: November 2003

Thrombopoietin, the ligand for the c‐mpl receptor, promotes proliferation and maturation of megakaryocytes. An ELISA using a chimaeric receptor, mpl‐IgG, for capture, and rabbit antibody to thrombopoietin for detection was developed for the quantitation of thrombopoietin in human serum or plasma. This ELISA preferentially detects full‐length thrombopoietin compared to the bioactive N‐terminal half of the molecule which has homology to erythropoietin. Thrombopoietin was not detected (<0.16 ng/ml) in 88/89 healthy individuals. However, elevated thrombopoietin concentrations of up to 3 ng/ml were detected in 59/63 thrombocytopenic patients, including cancer patients following chemotherapy. In cancer patients receiving chemotherapy with (n = 12) or without (n = 6) peripheral blood progenitor cell transplantation, thrombopoietin concentrations varied inversely with platelet counts throughout the treatment period. In general, patients who received myeloablative chemotherapy on days −7 to −2 and peripheral blood progenitor cell transplantation on day 0 had high thrombopoietin levels (0.6–2.9 ng/ml) around day 5. Low platelet counts (<20 × 109/l) occurred between days 4 and 9. Patients who received high‐dose chemotherapy on day 1 (equivalent to day −7 for transplantation patients) to day 6 without transplantation had high thrombopoietin concentrations (1.4–2.3 ng/ml) around day 13 and low platelet counts occurred between days 7 and 17.
Thrombomodulin activity on mesothelial cells: perspectives for mesothelial cells as an alternative for endothelial cells for cell seeding on vascular grafts
Authors: Verhagen H. J. M. , Heijnen‐Snyder G. J. , Pronk A. , Vroom Th. M. , Van Vroonhoven Th. J. M. V. , Eikelboom B. C. , Sixma J. J. , De Groot Ph. G.
Published: November 2003

Lining the luminal surface of prosthetic small diameter bypasses with endothelial cells (EC) will lower its thrombogenicity. Unfortunately, human EC are scarce. Mesothelial cells (MC) may be a valuable alternative for EC, since they are abundantly available and have antithrombotic and fibrinolytic properties. An important anticoagulant function of EC is due to thrombomodulin (TM) on the surface. The presence of TM on omentally derived human MC is not known but would increase the chance of successful use of MC for cell seeding procedures.

The expression and localization of TM on human MC was studied using monoclonal antibodies. TM activity on cultured MC, and the influence of cytokines, was measured by the gen‐eration of activated protein C (APC), and was compared to EC.

TM is expressed on the surface of MC as well as intracellularly, both in vivo and in vitro. The TM‐dependent generation of APC was significantly higher on cultured MC than on cultured EC (817 ± 141 pMv 262 ± 38 pM; P<0.001); their reaction to cytokines was almost identical. Seeding the MC onto vascular prostheses did not change the TM activity.

Thrombomodulin is present and highly active on cultured and seeded MC. This may have major implications for MC as a source for cell seeding on vascular prostheses.
Anaplastic large cell (CD30/Ki‐1+) lymphoma in HIV+ patients: clinical and pathological findings in a group of ten patients
Authors: Nosari Annamaria , Cantoni Silvia , Oreste Pierluigi , Schiantarelli Clara , Landonio Giuseppe , Alexiadis Spyridon , Gargantini Livio , Caggese Liliana , Gambacorta Marcello , Morra Enrica
Published: November 2003

We compared the clinical and pathological features of 10 HIV+ CD30+ anaplastic large cell lymphoma (ALCL) patients with 28 HIV+ CD30+ non‐Hodgkin's lymphoma (NHL) patients. The incidence of ALCL among 38 HIV+ systemic NHL patients was 26%. Clinical features were similar in all the HIV‐related NHL cases, but ALCL patients seemed to differ from HIV+ CD30− systemic NHL only in the greater frequency of lung tumours (40% v 21%) without concomitant mediastinal mass, bone marrow (75% v 18%) and gastroenteric involvement (40% v 25%). Among the HIV+ ALCL patients, histologic subtypes did not differ in frequency from ALCL in the general population. The B phenotype was predominant (50%) as in other HIV‐related NHL. EBV genoma, studied in all HIV+ ALCL patients, was present in 3/10 by in situ hybridization (ISH) and in 5/10 cases using PCR. The clinical course of lymphomas was similar in CD30 positive and negative NHL patients. Overall survival also was short in our series, particularly in HIV+ ALCL (84 v 188 d), probably because of profound immunodepression of the ALCL patients.

Our findings suggest that severe immunodepression due to HIV infection determines — more than any other factor — the clinical features of HIV+ ALCL, making them very similar to those of other high‐grade systemic HIV+ NHL.
Soluble CD16 (sCD16), a marker of malignancy in individuals with monoclonal gammopathy of undetermined significance (MGUS)
Authors: MATHIOT C. , MARY J. Y. , TARTOUR E. , FACON T. , MONCONDUIT M. , GROSBOIS B. , POLLET J. P. , MICHAUX J. L. , Ziegler L. EULLER , SAUTÈS C. , BATAILLE R. , FRIDMAN W. H.
Published: November 2003

There are no well‐defined host markers to determine which patients with a diagnosis of monoclonal gammopathy of undetermined significance (MGUS) will progress to multiple myeloma (MM). In this preliminary study we measured plasmatic soluble Fcγ receptor type III (sFcγRIII or sCD16) in 54 individuals with MGUS, 35 patients with multiple myeloma (MM) and 29 healthy controls. We confirmed, through receiver operating characteristic (ROC) curve analysis, that a low level of sCD16 discriminates MM patients from controls. Indeed, for a sCD16 value of 1.3 μg/ml, the sensitivity, as well as the specificity, of this discrimination were both equal to 83%, i.e. 83% of MM patients had a plasmatic sCD16 value <1.3 μg/ml compared with only 17% of controls. Moreover, ROC curve analysis showed that a low sCD16 level also identifies among MGUS patients a subgroup of patients who rapidly progress towards multiple myeloma: in this comparison, for a sCD16 level of 1.3 μg/ml, sensitivity and specificity were 70% and 79% respectively. Therefore a low sCD16 level in MGUS indicated a high likelihood of rapid evolution to MM. In contrast to sCD16, soluble IL‐6R did not appear to be discriminant in this study.
A diagnostic test for heparin‐induced thrombocytopenia: detection of platelet microparticles using flow cytometry
Authors: Lee David H. , Warkentin Theodore E. , Denomme Gregory A. , Hayward Catherine P. M. , Kelton John G.
Published: November 2003

Based on our previous observation that heparin‐induced thrombocytopenia (HIT) sera can generate platelet microparticles from washed platelets in a heparin‐dependent fashion, we developed a test for HIT using flow‐cytometry to measure heparin‐dependent platelet microparticle formation. During the developmental phase of the assay the optimal physical conditions for microparticle generation were defined. 133 sera were then evaluated using the microparticle assay and the serotonin release assay to determine the threshold for defining a positive result that gave optimal sensitivity and specificity. The microparticle assay was then prospectively evaluated against the serotonin release assay in 202 sera referred to our laboratory for HIT testing. Overall agreement between the two assays was 96% (Cohen‘s kappa = 0.91). When the clinical data were reviewed on patients whose sera gave discrepant results between the two assays, no case of HIT was detected by one assay and missed by the other. The platelet microparticle assay is as accurate as the serotonin release assay and may be a useful non‐radioactive test for HIT.
Diclofenac‐induced immune haemolytic anaemia: simultaneous occurrence of red blood cell autoantibodies and drug‐dependent antibodies
Authors: Salama Abdulgabar , Kroll Hartmut , Wittmann Georg , Mueller‐Eckhardt Christian
Published: November 2003

During the last 5 years we have identified a total of 17 patients (nine females and eight males aged between 53 and 85 years) with immune haemolytic anaemia related to diclofenac (a nonsteroidal anti‐inflammatory drug). All patients developed acute intravascular haemolysis. Two patients died, and eight patients developed temporary renal failure that required haemodialysis. The direct antiglobulin test was positive with anti‐IgG and anti‐C3d in all cases, with anti‐IgA in 4/10 cases tested, and negative with anti‐IgM. The indirect antiglobulin test was moderately or weakly positive in 11 cases, and IgG autoantibodies could be eluted from the red blood cells (RBCs) of all patients. Initially, the diagnosis of autoimmune haemolytic anaemia of warm type was suggested in all cases. All patients had simultaneously developed autoantibodies and drug‐dependent antibodies. The majority of drug‐dependent antibodies (n=13) reacted with urine containing the drug and its metabolites (ex vivo antigen), the native drug, and diclofenac‐treated RBCs. The antibodies in the remaining four cases were detectable only in the presence of ex vivo antigen. Diclofenac appears to bind only weakly to RBCs in the absence of the drug‐dependent antibodies.

We conclude that diclofenac forms neoantigens with RBCs that may stimulate the production of autoantibodies and drug‐dependent antibodies. The resulting haemolytic syndrome is very similar to autoimmune haemolytic anaemia of warm type.
Model analysis of the contrasting effects of GM‐CSF and G‐CSF treatment on peripheral blood neutrophils observed in three patients with childhood‐onset cyclic neutropenia
Authors: SCHMITZ STEPHAN , FRANKE HORST , LOEFFLER MARKUS , WICHMANN H. ERICH , DIEHL VOLKER
Published: November 2003

Human cyclic neutropenia (CN) is a rare haematological disorder characterized by regular fluctuation in the serial count of blood neutrophils. The oscillations occur at subnormal levels with a stable 3‐week period. To reduce the risk of serious infections during the severe neutropenic nadir phases (<0.25 ×109 neutrophils/l) patients are usually treated with recombinant growth factors. Compared with G‐CSF, which has been shown to enhance the amplitudes substantially, the response to GM‐CSF is poor: neutrophil numbers are not amplified, the cycles remain unchanged or are dampened. However, two cases with a modest neutrophil increase have been reported in the literature. In a recently published clinical study the different effects of GM‐CSF and G‐CSF application have been investigated in the same patients. Based on a mathematical model of CN we previously proposed, the detailed neutrophil data measured in this study are analysed by simulation. The contrasting clinical results can be quantitatively explained by the model concept of regulatory control together with possible individual feedback defects, i.e. abnormally reduced mitotic responsiveness of granulopoietic progenitor cells to GM‐CSF and G‐CSF.
Extended multidrug resistance in haemopoietic cells
Authors: Marks Denese C. , Su Gloria M. I. , Davey Ross A. , Davey Mary W.
Published: November 2003

The development of drug resistance was studied in a series of haemopoietic cells to determine its relationship to cell lineage. Treatment of the U937 monocytic cell line with epirubicin (15 ng/ml) or vinblastine (8 ng/ml) induced drug‐resistant sublines with cross‐resistance to epirubicin (8‐ and 16‐fold respectively), vinblastine (5‐ and 20‐fold), paclitaxel (15‐and 42‐fold) and etoposide (19‐ and 13‐fold). However, sublines were also 3–5‐fold resistant to the alkylating agent chlorambucil, cis‐platinum and methotrexate, demonstrating an extended multidrug resistance (MDR) phenotype. These cells over‐expressed P‐glycoprotein, but decreased drug accumulation was not restored in the presence of verapamil, suggesting that the P‐glycoprotein was not functional. Similar drug treatment of the HL60 promyelocytic cell line also produced sublines exhibiting an extended MDR phenotype. The KG1a and the HEL cell lines expressed functional P‐glycoprotein and were resistant to the drug concentrations used for treatment. Multidrug resistance as mediated by P‐glycoprotein cannot explain the resistance of CML patients to chemotherapy, especially in blast crisis. The induction of an extended MDR phenotype specifically in myeloid cells in response to drug treatment may explain the resistance observed in the treatment of CML.
N‐Acetylglucosamine kinase and N‐acetylglucosamine 6‐phosphate deacetylase in normal human erythrocytes and Plasmodium falciparum
Authors: WEIDANZ JON A. , CAMPBELL PATRICK , MOORE DWIGHT , DELUCAS LAWRENCE J. , RODÉN LENNART , THOMPSON JERRY N. , VEZZA ANNE C.
Published: November 2003

The major pathways of glucose metabolism in the malaria parasite, Plasmodium falciparum, have now been elucidated, and the structures and properties of parasite‐specific enzymes are presently being investigated. Little is known, however, about the enzymes catalysing monosaccharide interconversions in the parasite. In the present investigation we have examined the pathway of N‐acetylglucosamine catabolism which, in higher organisms, involves the following reaction sequence: N‐acetylglucosamine → N‐acetylglucosamine 6‐phosphate → glucosamine 6‐phosphate →  fructose 6‐phosphate. Assay of the specific kinase (E.C. 2.7.1.59) catalysing the phosphorylation of the sugar showed that the enzyme is present in Plasmodium extracts as well as in normal human erythrocytes; specific activities of 7.2 and 5.3 nmol/h/mg protein were measured for the parasite and erythrocyte extracts, respectively. n‐Acetylglucosamine 6‐phosphate deacetylase (E.C. 3.5.1.25), catalysing the second reaction, was also detected in both normal and Plasmodium‐infected erythrocytes. At 75% parasitaemia, the deacetylase activity was close to 3 times higher than that of normal control cells. The erythrocyte deacetylase was purified approximately 16 000‐fold by chromatography on DE52 cellulose, chromatofocusing, and size exclusion chromatography. Attempts to purify the parasite enzyme by the same procedures were unsuccessful due to loss of activity. A partially purified erythrocyte deacetylase preparation (eluted from DE52 cellulose) had a pH optimum of 7.5, a pI of 6.0, as indicated by chromatofocusing, and a Km of 29 μM. In conjunction with previous investigations, the present study indicated that all three enzymes required for N‐acetylglucosamine utilization are present in Plasmodium parasites as well as in normal erythrocytes.
Murine Marrow Stromal Response to Myelotoxic Agents in vitro
Authors: Hauser Simon P. , Udupa Kodetthoor B. , Lipschitz David A.
Published: November 2003

Previous studies have shown that the haemopoietically active murine long‐term bone marrow culture (LTBMC) is a useful model for studying drug‐induced suppression and recovery of myelopoiesis. We studied the effects on stromal morphology and stromal progenitors, assessed as colony forming unit‐fibroblasts (CFU‐F), of the addition of either the antimetabolite methotrexate (MTX) or the betalactam ceftazidime (CEF) to LTBMC. The examination of 500 μg/ml CEF‐treated cultures revealed a stroma that appeared empty, with modest reduction in total stromal counts, and significant decreases in fat‐containing and endothelial cells. In contrast, treatment with 1 μM MTX for 1 week initially caused minimal morphologic stromal changes, thereafter total stromal cell count as well as fibroblastoid, endothelial, fat containing and macrophage cells significantly increased. Haemopoiesis and the stroma recovered. Both CEF and MTX reversibly suppressed stromal progenitor cells in LTBMC. When added directly to CFU‐F cultures, the concentrations resulting in a 50% colony growth inhibition were 214 μg/ml for CEF and 375 nM for MTX. These results suggest that CEF, but not MTX, has direct toxic effects on the stroma of established LTBMC. Stromal cell increase following MTX treatment probably indicates a stromal response that may contribute to haemopoietic cell recovery.
Elevated serum interferon γ and interleukin‐6 in patients with necrotizing lymphadenitis (Kikuchi's disease)
Authors: Kubota Masaru , Tsukamoto Reiko , Kurokawa Keiji , Imai Tsuyoshi , Furusho Kenshi
Published: November 2003

We investigated serum levels of interferon α, interferon γ, tumour necrosis factor α, interleukin‐2 (IL‐2) and interleukin‐6 (IL‐6) in patients with necrotizing lymphadenitis (Kikuchi's disease) (NL). Four male patients, diagnosed as having NL following biopsy of the affected lymph nodes and by the clinical course, were included in this study. All four patients had higher than normal serum interferon γ and IL‐6 levels during the acute phase, which returned to normal levels during the convalescent phase. Interferon α, tumour necrosis factor α and IL‐2 were, however, within the normal ranges. Our findings indicate the possibility that interferon γ and IL‐6 may play an important role in the pathogenesis of NL.
Low response to high‐dose intravenous immunoglobulin in the treatment of acquired factor VIII inhibitor
Authors: Crenier Laurent , Ducobu Jean , Des Grottes Jean‐Marin , Cerny Jean , Delaunoit Christian , Capel Paul
Published: November 2003

Prednisone is the classic first‐line therapy to suppress an acquired factor VIII inhibitor and may achieve complete remission in about 30% of patients. More recently, promising results have been reported with high‐dose intravenous immunoglobulin (IVIg). However, after an extensive review of the literature, we found only three complete remissions (12%) among the 26 assessable patients treated by IVIg. These data are in agreement with the low response to IVIg that we experienced in our series of patients. This study suggests that steroids should still be preferred to IVIg, an expansive therapy, to suppress an acquired factor VIII inhibitor.
Prognostic significance of additional chromosome abnormalities in adult patients with Philadelphia chromosome positive acute lymphoblastic leukaemia
Authors: Rieder Harald , Ludwig Wolf‐Dieter , Gassmann Winfried , Maurer Jürgen , Janssen Johannes W. G. , Gökbuget Nicola , Schwartz Stefan , Thiel Eckhard , Löffler Helmut , Bartram Claus R. , Hoelzer Dieter , Fonatsch Christa
Published: November 2003

The clinical and biological significance of additional chromosome aberrations was investigated in a large series of 66 adult patients with Philadelphia (Ph) chromosome positive acute lymphoblastic leukaemia (ALL). Additional chromosome changes were observed in 71% of the cases. 9p abnormalities were identified in 26%, and monosomy 7 as well as hyperdiploid karyotypes >50 were both found in 17% of cases. 9p anomalies were characterized by a low complete remission (CR) rate (58%) and an extremely short median remission duration (MRD; 100 d). In patients with monosomy 7, the poor treatment outcome was confirmed (CR rate 55%; MRD 113 d). In contrast, all patients with hyperdiploid karyotypes >50 achieved CR, and the overall survival was superior to all other Ph‐positive ALL patients except those without additional chromosome aberrations. Exclusive rearrangement of the minor breakpoint cluster region of the BCR gene and lack of coexpression of myeloid‐associated antigens in cases with 9p anomalies as well as a high frequency of rearrangements of the major breakpoint cluster region of the BCR gene in patients with monosomy 7 (89%) further substantiated that additional chromosome aberrations may characterize distinct subgroups of Ph‐positive ALL. Moreover, the necessity of the complementing use of chromosome banding analyses, polymerase chain reaction (PCR) assays, and fluorescence in situ hybridizations in the accurate identification of Ph‐positive patients has become evident due to variant Ph translocations in 3%, and negative PCR assays in 4% of the cases.
Long‐term stromal cultures produce dendritic‐like cells
Authors: Ni Keping , O'Neill H. C.
Published: November 2003

A stromal cell‐dependent long‐term culture (LTC) system has been developed from spleen which continuously generates non‐adherent cells with dendritic cell (DC) characteristics. Bioassays using factor‐dependent cell lines have revealed that both spleen and thymic stromal cultures secrete interleukin (IL)‐3 and IL‐6‐like growth factors. Conditioned medium from LTC also contains factors which appear to be unrelated to IL‐3 and induces growth of stromal cells from bone marrow. Non‐adherent cells generated in LTC were not T or B lymphoid cells or granulocytes, nor were mast cells detectable. Morphological and electron microscopic examination has also excluded the presence of macrophages (Mø). Cells with DC morphology have been detected by both light and electron microscopy. The majority of cells in the non‐adherent population were found to have multiple membrane pseudopodia, with a small acentric nucleus. These appear to be the precursors of DC. They expressed cell surface markers detectable with DC‐specific antibodies and antibody specific for major histocompatibility complex Class II molecules. A proportion of cells also expressed myeloid markers, but since this expression was not supported by histochemical staining for myeloperoxidase or non‐specific esterase, it was concluded that the cells produced are not typical of the myeloid lineage. Cells generated in LTC were shown to be potent stimulators for allogeneic and syngeneic MLR and for antigen‐specific T‐cell proliferation.
Biological properties of peripheral blood progenitor cells mobilized by cyclophosphamide and granulocyte colony‐stimulating factor
Authors: Scott M. A. , Apperley J. F. , Bloxham D. M. , Jestice H. K. , John S. , Marcus R. E. , Gordon M. Y.
Published: November 2003

Patients transplanted with mobilized blood progenitor cells (PBPC) recover their neutrophil counts more rapidly than patients transplanted with bone marrow even when they receive the same dose/kg of granulocyte‐macrophage colony‐forming cells (CFU‐GM). Here we have sought a biological explanation for this phenomenon. Most CD34‐positive PBPC are quiescent (<1% in S phase) when they are collected from the bloodstream of patients treated with cyclophosphamide and granulocyte colony‐stimulating factor (G‐CSF), but we have shown that they are able to resume proliferation rapidly in vitro by measuring the kinetics of CFU‐GM production by primitive plastic‐adherent (PΔ) cells. Also, PΔcells in PBPC harvests, unlike normal marrow PΔ cells, were insensitive to cell‐cycle restraint imposed by contact with marrow‐derived stromal cells. We found that PΔ cells in PBPC collections produce relatively more CFU‐GM and relatively fewer BFU‐E than PΔ cells in bone marrow, indicating that granulopoiesis might occur at the expense of erythropoiesis, but we were unable to find any differences in the kinetics of granulocytic maturation between PBPC and bone marrow. Our interpretation of these findings is that transplanted PBPC rapidly enter the cell cycle and contact with stromal cells in the marrow does not reduce the proportion of progenitors participating in neutrophil production. Consequently, neutrophil recovery after PBPC infusion is more rapid than neutrophil recovery after marrow infusion. Granulopoiesis at the expense of erythropoiesis may also contribute to this effect.
Comparative analysis of the impact of risk profile and of drug therapy on survival in CML using Sokal's index and a new score
Authors: Hehlmann R. , Ansari H. , Hasford J. , Heimpel H. , Hossfeld D. K. , Kolb H. J. , Löffler H. , Pralle H. , Queisser W. , Reiter A. , Hochhaus A.
Published: November 2003

Survival times in chronic myeloid leukaemia (CML) may vary widely depending on the risk profiles of patients. This fact is frequently not, or not sufficiently, considered in evaluating survival in CML, and some studies do not report risk profiles. Therefore we analysed the relative impact of risk profile and therapy on survival using the median survival times of therapy groups and of risk groups of the three‐arm randomized German CML Study I (interferon alpha v hydroxyurea v busulphan; median survival times 65 v 56 v 45 months, n=490, median observation time 70.4 months). The impact of risk profile (Sokal) on survival as determined by the survival difference between high and low risk patients (40 months) was twice the maximum survival difference between treatment groups (20 months). A similar ratio was obtained after stratification for therapy and for risk profile. Since Sokal's index has been reported to prognostically discriminate IFN‐treated patients less well than chemotherapy‐treated patients, a new score with better discrimination of IFN‐treated patients was also used. The results were similar for both scores. We conclude that the risk profile at diagnosis is still more important for survival of CML patients than therapy. Therefore patients should be stratified according to risk profile for comparisons of survival times between studies and treatment arms.
CD40 and CD95 induce programmed cell death in the human myeloma cell line XG2
Authors: Bergamo Alberta , Bataille RÉgis , Pellat‐Deceunynck Catherine
Published: November 2003

We have previously demonstrated that CD40 stimulation induced a cellular growth arrest of the highly CD40‐positive myeloma cell line XG2. To further characterize this inhibition of proliferation, we looked for a possible induction of apoptosis. Since no DNA fragmentation could be detected, we used newly described techniques that enable detection of apoptosis independently of DNA degradation, i.e. supravital exposure to propidium iodide (PI) and Annexin V labelling. We demonstrated that CD40 effectively induced programmed cell death. Furthermore, we have shown that CD95 (Fas) stimulation significantly enhanced the CD40‐induced apoptosis.