bluuebunny/crossref_metadata_2025 huggingface dataset:
Detection of monoclonal B lymphocytes in bone marrow and peripheral blood of multiple myeloma patients by immunoglobulin gene rearrangement studies
Authors: Van Riet Ivan , Heirman Carlo , Lacor Patrick , Waele Marc De , Thielemans Kris , Van Camp Ben
Published: March 2008

To investigate whether B lymphocytes are involved in the malignant cell clone of multiple myeloma (MM), we performed immunoglobulin gene rearrangement analysis of mononuclear cells and separated B lymphocytes, isolated from bone marrow and peripheral blood of MM patients. The B lymphocytes were separated by immunomagnetic beads, coated with an HLA class II specific antibody. Southern blot analysis with a JH probe revealed in the bone marrow of three out of seven patients identical immunoglobulin gene rearrangements in the B lymphocytes when compared to the plasma cells. Out of 10 patients, two patients with a high tumour burden were found to have monoclonal B lymphocytes in the peripheral blood. These results suggest that B lymphocytes in the bone marrow are part of the myeloma clone and that they can circulate in the peripheral blood. Although previous studies indicated that the ratio of K to λ bearing lymphocytes in the peripheral blood can provide evidence for B cell monoclonality, we did not find a correlation between the results of K/λ analysis and immunoglobulin gene rearrangement.
Analysis of surface antigen expression of human immunoglobulin‐secreting cells: phenotypic heterogeneity in normal counterparts of myeloma cells
Authors: Tominaga Nobuhiko , Katagiri Shuichi , Ohnishi Mitsunobu , Nakao Hirohisa , Oritani Kenji , Yagura Hirosuke , Tamaki Toshiharu , Kanayama Yoshio , Yonezawa Takeshi , Tarui Seiichiro
Published: March 2008

Human myeloma cells are malignant counterparts of plasma cells which represent the most differentiated B cells. Myeloma cells are, however, heterogeneous in their surface antigen expression (Katagiri et al, 1984, 1985), which may reflect that normal plasma cells have a spectrum of differentiation. To test this hypothesis, immunoglobulinsecreting cells (ISC) of non‐neoplastic nature were studied with regard to their surface antigen expression by using a combination of reverse haemolytic plaque assay and complement‐dependent cytolysis. Non‐neoplastic ISC were found to have a broad spectrum of differentiation stages from the immature type of CD20+, HLA‐DR+, CD38+ in the peripheral blood to the mature type of CD20–, HLA‐DR–, CD38+ in the bone marrow. In patients with polyclonal B cell activation (PBA), ISC showed a more immature antigen expression in comparison with ISC in normal controls or patients without PBA. The surface antigen development of ISC was clearly demonstrated throughout the stages in the analysis of mitogen‐induced ISC in vitro. No significant difference in the surface phenotype of ISC was found among heavy chain classes. Thus, non‐neoplastic ISC show a spectrum of differentiation similar to that of myeloma cells, depending on the site where ISC are located, and on the degree of PBA in vivo.
Biological and clinical features of acute lymphoblastic leukaemia with cytoplasmic granules or inclusions: description of eight cases
Authors: Cantù‐Rajnoldi Angelo , Invernizzi Rosangela , Biondi Andrea , Banfi Patrizio , Zoia Adele , Fazio Pasqualina De , Polli Nicoletta
Published: March 2008

We describe eight patients (four children and four adults) with an acute lymphoblastic leukaemia (ALL) with cytoplasmic granules or inclusions. The incidence of this variant of acute leukaemia in our whole series of patients with ALL is 1.8%. The granules or inclusions were usually positive for aspecific esterases (ANAE) and/or acid phosphatase, and the immunophenotype was in all cases typical of a CALLA positive B‐lineage ALL (CD10+, CD19+ and/or CD24+, DR+, TdT+, anti‐T‐, anti‐My‐, SIg‐). In one paediatric case, CD33 was unusually coexpressed. Ultrastructural investigations were performed in one case and demonstrated large granules containing vesicles, usually membrane bound, in the majority of blast cells. In the two cases analysed, Ig heavy chain gene rearrangement was detected. In this series of patients prognosis was poor since three never achieved a complete remission, four relapsed and only one is still in first continuous remission.
Long‐term results of the CHOP regimen in stage C chronic lymphocytic leukaemia
Authors:   French Cooperative Groupon Chronic Lymphocytic Leukaemia
Published: March 2008

This paper reports the results of the third interim analysis of a randomized clinical trial in poor prognosis chronic lymphocytic leukaemia (stage C). From May 1980 to February 1985, 70 stage C patients were randomized between the two polychemotherapies COP and CHOP (COP+doxorubicin). At the reference date of 1 June 1987 used for this analysis, the mean follow‐up was 58 months, 46 deaths were observed, 27 in the COP group and 19 in the CHOP group. Overall survival was significantly improved in the CHOP group (P= 0.0005). The 3‐year survival rates were 71% in the CHOP group and 28% in the COP group and the median survival times were 62 months in the CHOP group as compared to 22 months in the COP group. This benefit with CHOP was confirmed when adjusting treatment comparison for nine parameters (P= 0.001) and when considering other endpoints than survival, namely 9‐month status and stage. This third interim analysis confirms the results of the first interim analysis that led to an early stopping of this clinical trial and clearly indicates the beneficial effect of low doses of doxorubicin in advanced chronic lymphocytic leukaemia.
Cytokine activity after human bone marrow transplantation III. DEFECT IN IL2 PRODUCTION BY PERIPHERAL BLOOD MONONUCLEAR CELLS IS NOT CORRECTED BY STIMULATION WITH Ca++ IONOPHORE PLUS PHORBOL ESTER
Authors: Cooley Margaret A. , McLachlan Karen , Atkinson Kerry
Published: March 2008

Previous studies have shown that interleukin 2 (IL2) production by peripheral blood mononuclear cells (PBMC) is severely impaired post allogeneic bone marrow transplantation, whereas production of interferon‐γ (IFN‐γ) is at most marginally depressed. To investigate the mechanisms behind this apparently differential inhibition of lymphokine production, we stimulated PBMC from recipients of HLA‐identical sibling bone marrow transplants with phytohaemagglutinin (PHA), PHA + phorbol ester (PMA) (to bypass accessory cell requirements) or Ca++ ionophore + PMA (to bypass both accessory cell and T cell surface receptor (CD2 and/or CD3/Ti interactions). Increasing the potency of the stimulus increased the amount of IL2 and IFN produced by PBMC from both normal volunteers and from marrow transplant recipients, but for each stimulus the amount of IL2 produced by marrow transplant recipient PBMC remained 10–100‐fold lower than that produced by normal PBMC, suggesting an underlying defect in IL2 production by marrow transplant recipient T cells, not due to accessory cell or CD2 defects. Selection experiments showed that CD3+ cells were the primary IL2 producers, and we were unable to demonstrate presence of suppressor cells in marrow transplant PBMC. Statistical analysis of the clinical factors possibly affecting lymphokine synthesis showed that in vivo cyclosporin A did not affect the in vitro capacity of PBMC to produce cytokines, although steroid therapy had a negative effect on IL2 production. The only variable significantly affecting IL2 and IFN production in marrow transplant recipients was increasing time post transplant. It is suggested that the defect in IL2 but not IFN production could be due to either a selective reduction in the frequency of IL2 producing cells as opposed to IFN producing cells, or to a reduction in the amount of IL2 produced per cell in marrow transplant recipients.
Autoantibodies to platelet glycoproteins in patients with disease‐related immune thrombocytopenia
Authors: Berchtold Peter , Harris Jeffrey P. , Tani Patricia , Piro Lawrence , McMillan Robert
Published: March 2008

Although increased platelet destruction and elevated platelet‐associated IgG have been shown in patients with lymphomas and various autoimmune diseases, such as systemic lupus erythematosus (SLE), there have been few studies evaluating autoantibodies against platelet‐specific antigens. We evaluated 24 patients retrospectively with disease‐related thrombocytopenia (12 with lymphoproliferative diseases and 12 with various autoimmune disorders) using a recently reported antigen‐specific assay.

Autoantibodies against platelet GPIIb/IIIa or GPIb/IX were noted in 15 of the 24 patients (10 of 12 with autoimmune disease and five of 12 with lymphoproliferative disorders). Platelet‐associated autoantibodies were present in 60% and plasma autoantibodies in 33%. Anti‐GPIIb/IIIa autoantibodies were much more common than those against GPIb/IX.

In one patient each with thrombocytopenia and either SLE or myasthenia gravis, absorption of plasma with platelets completely removed the anti‐GPIIb/IIIa autoantibodies, but did not affect the level of anti‐cochlear autoantibody involved with immune‐mediated hearing loss in the SLE patient or the anti‐acetylcholine receptor autoantibody in the myasthenic patient.

These findings show that, in some cases of disease‐related immune thrombocytopenia, autoantibodies against GPIIb/IIIa or GPIb/IX can be detected similar to those seen in chronic ITP. As shown in two patients with multiple autoimmune manifestations, the various autoantibodies have diverse specificities and do not crossreact.
A spectrophotometric method for the assay of pyrimidine 5′‐nucleotidase in human erythrocytes
Authors: Amici Adolfo , Natalini Paolo , Ruggieri Silverio , Vita Alberto , Magni Giulio
Published: March 2008

A new spectrophotometric assay for human erythrocyte pyrimidine 5′‐nucleotidase using a continuous spectrophotometric method is described. The activity is detected by measuring uracil, which is produced by a coupled assay including cytidine deaminase and uridine nucleosidase. The results obtained with the continuous spectrophoto‐metric method have been confirmed by high pressure liquid chromatography (HPLC) analysis. This procedure, not based upon inorganic phosphate formation and avoiding the use of radioactive material, offers significant advantages with respect to commonly adopted assays.
Interleukin 3 alone does not support the proliferation of bone marrow cells from A/J mice: a novel system for studying the synergistic activities of IL‐3
Authors: Morris C. F. , Salisbury J. , Kobayashi M. , Townsend P. V. , Hapel A. J.
Published: March 2008

The number of colonies produced by bone marrow cells in response to interleukin 3 (IL‐3) in soft agar cultures varies according to the strain of the donor mice. A/J, AKR, A.TH and A.TL bone marrow cells are particularly hyporesponsive, producing only occasional colonies in the presence of IL‐3. Bone marrow cells from all strains of mice, including A/J, produce distinctively large colonies in response to the combination of IL‐3 and macrophage colony stimulating factor (M‐CSF). In cultures of A/J bone marrow cells, the synergy between IL‐3 and M‐CSF is further reflected in an increase in both the number and the variety of colonies produced. The increase in colony numbers may be due to the priming of a population of A/J colony‐forming‐cells (CFCs) by IL‐3, enabling them to respond to M‐CSF. In support of this notion, IL‐3 enhances the level of c‐fms (M‐CSF receptor) messenger RNA in cultures of A/J bone marrow cells. It is also possible that a subpopulation of CFCs requires both IL‐3 and M‐CSF as co‐mitogens. The A/J strain provides a novel system for studying the mechanisms involved in the interaction between IL‐3 and M‐CSF in haemopoiesis.
Platelet activation by CD9 monoclonal antibodies is mediated by the FCγII receptor
Authors: Worthington Ronald E. , Carroll Roger C. , Boucheix Claude
Published: March 2008

The function of the human cell surface CD9 antigen is not known, yet monoclonal antibodies (mAbs) of the IgG1 subclass in the CD9 cluster induce activation of platelets. Previously it had been shown that this activation pathway is comparable both in kinetics and extent to physiological agonists such as thrombin. Here it is demonstrated that activation with CD9 mAbs depends on interaction of the Fc part of the CD9 antibody molecule with Fc receptors on the platelet surface, since: (i) mAb directed against the Fc receptor totally blocked the platelet response to CD9 mAb; and (ii) F(ab')2 fragments of the CD9 mAb SYB‐1 which bound to platelets, as demonstrated by flow cytometry, failed to activate them. Furthermore, platelet activation by CD9 mAb closely paralleled the activation caused by crosslinking Fc receptors when comparing: (i) kinetics and extent of aggregation; (ii) thromboxane synthesis; (iii) calcium flux: and (iv) the cytoplasmic alkalinization response. Thus it is concluded that CD9 antigen itself does not necessarily participate in stimulus‐response coupling leading to platelet activation by CD9 mAbs, and that this activation can be entirely accounted for by the Fc receptor pathway mechanism. The results suggest a possible novel mechanism for platelet consumption in cases of immune thrombocytopenia.
In vitro suppressor T lymphocyte dysfunction in autoimmune thrombocytopenic purpura associated with a complement‐fixing antibody
Authors: Hymes Kenneth B. , Karpatkin Simon
Published: March 2008

Ig secretion of peripheral blood mononuclear cells (PBMC) was measured in Epstein Barr virus (EBV) seropositive autoimmune thrombocytopenic purpura (ATP) patients and controls following in vitro infection with EBV. EBV infected PBMC from patients with ATP secreted Ig for a longer period of time than EBV infected control cells and had higher peak Ig production. Removal of T cells or CD8 cells from control PBMC increased the duration and level of Ig production to that achieved by ATP PBMC. Total T or CDS cell depletion of ATP PBMC had no effect on the enhanced level or duration of Ig secretion. Depletion of control CD4 lymphocytes decreased Ig production.

Treatment of EBV infected control PBMC with either ATP sera or purified IgG (plus complement) increased the duration and level of production of Ig to that observed in ATP PBMC, control PBMC depleted of all T cells or control PBMC depleted of CD8 cells. This antibody effect could be reversed by reconstitution with control T cells. Thus, patients with ATP produce an antibody which leads to suppressor cell dysfunction. Defective function of these cells may lead to a disorder of immune regulation in which autoantibodies are produced against platelets.
Long‐term bone marrow damage after chemotherapy for acute myeloid leukaemia does not improve with time
Authors: Chang J. , Geary C. G. , Testa Nydia G.
Published: March 2008

Thirteen patients with acute myeloid leukaemia (AML) who had been in unmaintained complete remission for at least 5 years following successful chemotherapy, were studied by marrow culture techniques regularly for periods of up to 5 years. In 10 patients, short‐term clonal assay revealed the incidence of granulocyte‐macrophage progenitor cells (GM‐CFC) to be at the lower limit of the normal range; but three showed wide fluctuations in GM‐CFC numbers. Moreover the generation of GM‐CFC in long‐term bone marrow culture (LTBMC) was impaired in eight of nine evaluable patients. These results suggest that, despite normal blood counts, impairment of haemopoiesis persists in patients who are long‐term survivors of AML, and this does not improve with the passage of time.
HLA class I expression on erythrocytes and platelets from patients with systemic lupus erythematosus, rheumatoid arthritis and from normal subjects
Authors: Botto Marina , So Alex K.‐L. , Giles Carolyn M. , Mason Philip D. , Walport Mark J.
Published: March 2008

It has previously been shown, by a haemagglutination assay, that patients with systemic lupus erythematosus (SLE) express increased levels of HLA class I on erythrocytes compared with normal subjects and patients with rheumatoid arthritis (RA). A radioligand‐binding assay, using monoclonal antibody W6/32, was devised to quantify HLA class I expression on erythrocytes and platelets. An increased number of class I molecules was expressed on erythrocytes from 45 patients with SLE (mean = 354 molecules per cell, median = 255 molecules, range = 30–1270 molecules per cell), compared with cells from 46 normal subjects (mean = 132, median = 78, range = 40–550) and 31 RA patients (mean = 132, median = 89, range = 26–497). The presence of HLA‐B7 correlated with increased class I expression on erythrocytes from both normal subjects and patients with SLE. Levels of HLA class I in serum were measured. All subjects with HLA‐A9 (A23, 24) showed higher levels of serum class I than their A9‐negative counterparts, and there was no difference in levels between SLE patients and normal subjects. There were no correlations between class I levels in serum and on erythrocytes amongst SLE patients or normal subjects. Red cells were fractionated, according to their age in vivo, on Percoll gradients. Class I levels fell with increasing erythrocyte age in all individuals, but were higher in all fractions from SLE patients compared with age‐matched fractions from normal subjects. HLA‐B7‐positive erythrocytes also expressed higher class I levels in each Percoll fraction, compared with their HLA‐B7‐negative counterparts, suggesting that enhanced B7 expression is not due to greater structural stability of this class I allotype. These data are compatible with the hypothesis that class I is expressed as an intrinsic protein of erythrocyte membranes and that expression is increased amongst patients with SLE.
Thrombocytopenia after bone marrow transplantation for leukaemia: changes in megakaryocyte growth and growth‐promoting activity
Authors: Adams J. A. , Gordon A. A. , Jiang Y. Z. , Macdonald D. , McCarthy D. M. , Zuiable A. , Treleaven J. , Powles R. L. , Barrett A. J.
Published: March 2008

Megakaryocyte growth‐promoting activity (MK‐GPA) was scored on a scale of 0‐3 in the serum of 23 patients up to 120 d following bone marrow transplantation (BMT) for leukaemia. Nine of] 9 allografts and two of four autografts had thrombocytopenia requiring platelet transfusion more than 30 d after BMT. There was a close correlation between MK‐GPA and platelet count. MK‐GPA reached a maximum before day 30 after BMT but remained elevated in patients with persisting thrombocytopenia secondary to poor engraftment, graft‐versus‐host disease (GVHD) or relapse. Recent platelet transfusion did not suppress serum MK‐GPA. Two of four patients undergoing autologous BMT for acute myeloid leukaemia (AML) showed delayed platelet recovery and persistence of MK‐GPA in the serum. Seven further AML remission marrows were tested for megakaryocyte production before or after autologous BMT, using pooled sera with known MK‐GPA activity. Megakaryocyte generation was reduced before BMT and absent in post transplant samples. This failure of MK production was not corrected by T‐cell depletion or by the presence of adherent cells from normal marrow. We conclude that thrombocytopenia after BMT is associated with an appropriate increase in MK‐GPA levels in response to a reduction in the megakaryocyte pool rather than the platelet pool, and that persisting thrombocytopenia after autologous BMT is due to decreased numbers of available megakaryocyte precursors.
Effects of recombinant human erythropoietin on anaemic W/Wv and SI/SId mice
Authors: Cynshi Osamu , Satoh Koh , Higuchi Masato , Imai Nobuo , Kawaguchi Tsutomu , Hirashima Kunitake
Published: March 2008

Summary. The effects of recombinant human erythropoietin (rHuEPO) on anaemic W/Wv and SI/SId mice were investigated. rHuEPO was injected every day for a week in doses up to 86000 iu/kg. Wv/+ and SId+ mice, which have genetically a weak anaemia, received 17 or 86 iu/kg of rHuEPO and showed dose‐dependent increases in haemoglobin, PCV, RBC and reticulocytes to the same extent as that in normal mice. W/Wv mice also showed increases in the haematological parameters in response to 8600 iu/kg of rHuEPO but the dose was much higher than that for normal mice. A reticulocyte increase in W/Wv mice appeared later than in normal mice and was not sustained for 2 weeks even though the rHuEPO treatment was continued. SI/SId mice, however, did not show any significant haematological effect from doses up to 86 000 iu/kg. In both W/Wv and SI/SId mice receiving 8600 and 86000 iu/kg of rHuEPO, respectively, an increase in splenic or bone marrow CFU‐E was observed regardless of the defect in their haemopoietic systems. The plasma erythropoietin (EPO) level in W/Wv and SI/SId mice was inversely correlated with the haemoglobin, indicating that EPO production was not influenced by the haemopoietic defect and was regulated by the hypoxic properties of the anaemia. These results indicate that a large dose of exogenous rHuEPO is effective for the anaemia in W/Wv mice caused by a stem cell defect but not for the anaemia in SI/SId mice caused by a defective microenvironment.
Transfusion of ABO‐mismatched platelets leads to early platelet refractoriness
Authors: Carr Robert , Hutton Jane L. , Jenkins Jill A. , Lucas Geoffrey F. , Amphlett Nigel W.
Published: March 2008

Summary. Forty‐three consecutive patients previously unexposed to platelets and undergoing treatment for acute leukaemia or autografting for relapsed Hodgkin's lymphoma were randomized to receive transfused platelets of either their own ABO group (OG) or of a major mismatched group (MMG). The 26 evaluable patients were equally distributed between the two study groups.

Nine of 13 (69%) MMG patients became refractory with a median onset at transfusion 7 (15 d), compared with only one of 13 (8%) OG patients (P=0.001). Refractoriness was associated with the formation of high titre isoagglutinins, anti‐HLA and platelet specific antibodies. In one patient refractoriness appeared to be due to high titre isoagglutinins alone. Six other patients developed an increase in isoagglutinin titre sufficient to adversely affect platelet increments. Patients receiving ABO‐mismatched platelets had a higher incidence of anti‐HLA antibodies (5 v. 1) and platelet specific antibodies (4 v. 1). ABO‐mismatched platelets transfused prior to the onset of refractoriness resulted in increments similar to those achieved by ABO‐matched platelets.

The study demonstrates that ABO‐mismatched platelets are as effective as matched platelets in patients with low titre isoagglutinins requiring only few transfusions. However, the greater incidence of early refractoriness induced in MMG patients indicates that ABO‐mismatched platelets should not be given to patients with marrow failure requiring long‐term support.
A haemolytic syndrome associated with the complete absence of red cell membrane protein 4.2 in two Tunisian siblings
Authors: Ghanem A. , Pothier B. , Marechal J. , Ducluzeau M. T. , Morle L. , Alloisio N. , Feo C. , Abdeladhim A. Ben , Fattoum S. , Delaunay J.
Published: March 2008

Summary. We report on the complete absence of protein 4.2 in two Tunisian siblings. The propositus presented with a haemolytic anaemia that evolved in an intermittent fashion until she was cured by splenectomy. Her red cells had a normal morphology, as well as a normal deformability upon osmotic gradient ektacytometry. SDS‐polyacrylamide gel electrophoresis failed to reveal any protein 4.2. Using anti‐protein 4.2 polyclonal antibodies. Western blots were also unable to detect protein 4.2. Preparation of inside out vesicles resulted in no detectable loss of ankyrin. The propositus's sister presented with a haemolytic anaemia but had not undergone splenectomy; she showed the same biochemical features. The two cases presented of missing protein 4.2 are the first ones to be described outside the Japanese population. Considered as homozygotes for some defect that must alter the protein 4.2 gene itself, they exemplify a unique syndrome pertaining neither to elliptocytosis nor to spherocytosis, at least not closely. The parents, who are first cousins and whom we regarded as heterozygotes, were clinically and morphologically normal; they had a normal content of protein 4.2. Therefore, the 4.2 (–) haemolytic anaemia appears as entirely recessive.
The role of interleukin‐1 and tumour necrosis factor‐α in human multiple myeloma
Authors: Carter Anna , Merchav Shoshana , Silvian‐Draxler Ilana , Tatarsky Ilana
Published: March 2008

This study investigates the capacity of interleukin‐1α (IL‐1α), interleukin‐1β (IL‐1β) and tumour necrosis factor‐α (TNF‐α) to induce interleukin‐6 (IL‐6) production in freshly isolated myeloma cells (MC) and bone marrow‐derived stromal cells (MSC). Recombinant human (rh) IL‐1α, IL‐1β and TNF‐α augmented production of IL‐6 in human MC.IL‐6 was determined on a factor‐dependent Cess cell line. This activity was completely abrogated by anti‐IL‐6 antibodies. Prior incubation of IL‐1α, IL‐1β and TNF‐α with their respective antibodies inactivated the ability of recombinant cytokines to stimulate the release of IL‐6 from myeloma cells. IL‐1α, IL‐1β and TNF‐α enhanced 3H‐TdR uptake in myeloma cells through IL‐6, as antibodies to IL‐6 completely abolished the DNA synthesis induced by culture supernatants of MC exposed to these cytokines. rhIL‐6 reversed the inhibitory action of anti‐IL‐6 antibodies and reinduced DNA synthesis in MC. Next we found that IL‐lα, IL‐1β and TNF‐α induced MSC to produce IL‐6. In contrast, supernatants of unstimulated MSC did not contain detectable IL‐6 biologic activity. Further data demonstrated that human MC were able to induce IL‐6 production in MSC. The stimulatory activities of MC appeared to be mediated through endogenously released IL‐1, as the addition of antibodies towards IL‐1 at the initiation of cocultures completely abrogated the IL‐6 production. We conclude from our data that IL‐1 and TNF‐α may play an important role in the pathogenesis of human multiple myeloma.
Calpain proteolysis of von Willebrand factor enhances its binding to platelet membrane glycoprotein IIb/IIIa: an explanation for platelet aggregation in thrombotic thrombocytopenic purpura
Authors: Moore Jane C. , Murphy William G. , Kelton John G.
Published: March 2008

We have previously observed calpain activity (calcium‐dependent cysteine protease) in sera from patients with acute thrombotic thrombocytopenic purpura (TTP). The calpain activity was not present following recovery and was not detected in other thrombocytopenic disorders. We postulated that this enzyme could participate in the pathogenesis of TTP. Because other investigators have demonstrated abnormalities of von Willebrand factor (vWF) in patients with TTP, we proposed that calpain might interact with vWF in TTP. To challenge this hypothesis, we measured the binding of untreated and calpain‐treated vWF to normal and ADP or calpain activated platelets. Untreated vWF bound in a specific and saturable fashion to activated platelets, but only at low (30 μm) calcium concentrations. Von Willebrand factor did not bind to activated platelets at physiological (2 mm) calcium concentrations. Calpain proteolysis of vWF changed the binding characteristics of the vWF so that it had greatly increased binding to both ADP and calpain activated platelets. The calpain‐proteolysed vWF bound to activated platelets at both low and physiological calcium concentrations, and was capable of causing platelet aggregation. The calpain‐proteolysed vWF bound to the activated platelets via glycoproteins IIb/IIIa as demonstrated by inhibition studies using monoclonal antibodies against glycoproteins IIb/IIIa and Ib. It also had a high binding affinity and was capable of inhibiting the binding of radiolabelled fibrinogen to the activated platelets at physiological calcium concentrations. Calpain also proteolysed fibrinogen, but the calpain altered fibrinogen had normal platelet reactivity.

These studies provide further insight into the pathogenesis of the platelet aggregation of thrombotic thrombocytopenic purpura. Calpain proteolyses vWF and can produce the characteristic loss of large multimers seen on sodium dodecyl sulphate (SDS)‐agarose gel electrophoresis. The altered vWF is highly reactive with activated platelets and binds to platelet glycoproteins IIb/IIIa and participates in formation of the platelet aggregates that characterize this disease.
Post‐hepatitic aplastic anaemia in children in Taiwan, a hepatitis prevalent area
Authors: Liang Der‐Cherng , Lin Kai‐Hsin , Lin Dong‐Tsamn , Yang Chao‐Ping , Hung Kun‐Long , Lin Kuo‐Sin
Published: March 2008

Aplastic anaemia is a rare but serious complication of hepatitis, and hepatitis is an unusual cause of aplastic anaemia in children in the West. However, the relative frequencies of acquired aplastic anaemia in children in Taiwan, a hepatitis prevalent area, differ from those in the West, in the very high frequency of post‐hepatitic aplastic anaemia (23.9% of all cases of aplastic anaemia). This may account for the higher incidence of aplastic anaemia in children in Taiwan. Although the prognosis of post‐hepatitic severe aplastic anaemia was very poor, the present study using bone marrow transplantation, antithymocyte (or antilymphocyte) globulin, high‐dose methylprednisolone and cyclosporin, etc., has improved the response rate and the survival.
Erythropoietin production in kidney tubular cells
Authors: Maxwell Alexander P. , Lappin Terence R. J. , Johnston Colin F. , Bridges John M. , McGeown Mary G.
Published: March 2008

The erythropoietin gene has been cloned in three mammalian species including man and recombinant erythropoietin is now used to treat the anaemia of chronic renal failure. Despite the isolation of the gene the precise cellular location of erythropoietin synthesis remains controversial. We present studies which demonstrate erythropoietin production by kidney tubular cells. Erythropoietin gene expression (messenger RNA) was detected by in situ hybridization using an oligonucleotide gene probe and the translated protein product by immunohistochemistry employing antibodies raised to pure recombinant DNA derived erythropoietin.
Alpha‐2a‐interferon/melphalan/prednisone versus melphalan/prednisone in previously untreated patients with multiple myeloma
Authors: Montuoro Aldo , De Rosa Luca , De Blasio Angelo , Pacilli Leonardo , Petti Nicola , De Laurenzi Antonio
Published: March 2008

Alpha‐2a‐interferon (IFN) has demonstrable activity in advance and refractory multiple myeloma (MM), because of the in vitro synergism between IFNs and cytotoxic agents we report the preliminary results of a therapeutic trial of 50 patients with MM. Twenty‐eight patients were randomized to receive melphalan plus prednisone (MP) and 22 were randomized to receive IFN plus MP (IFN‐MP). Criteria for response, progression and relapse were those of the South‐western Oncology Group. 95% of the patients receiving IFN‐MP responded to therapy as opposed to 68% of the patients receiving MP (P<0·05). Response was independent of M‐component immunoglobulin class but in stage III it was higher in the IFN‐MP group than in the MP group (P<0·05). The combination IFN‐MP was well tolerated without unusual or unexpected toxic effects. The response duration time was longer in the IFN‐MP group than in the MP group (P<0·025). The median survival was 80 weeks in the MP group and in the IFN‐MP group the 93% of patients were still alive after 90 weeks (P<0·025). Our results show that the use of the IFN as an adjuvant to MP improves the percentage of responders, the response duration time and the median survival of untreated patients with MM.
Evidence for a von Willebrand factor defect in factor VIII binding in three members of a family previously misdiagnosed mild haemophilia A and haemophilia A carriers: consequences for therapy and genetic counselling
Authors: Mazurier Claudine , Gaucher Christine , Jorieux Sylvie , Parquet‐Gernez Armelle , Goudemand Maurice
Published: March 2008

A plasma von Willebrand factor (vWf) defect limited to its failure to bind factor VIII (FVIII) was previously characterized in a woman with FVIII deficiency and normal primary haemostasis. By usingin vitrotests we found a similar pattern in three siblings of another family previously thought to be affected with mild haemophilia A. Furthermore, a decrease in vWf ability to bind FVIII was found in the parents and the brother of the three patients. This decrease was consistent with heterozygous expression of a recessive vWf gene abnormality. FVIII deficiency was corrected by infusion with a vWf concentrate almost devoid of FVIII coagulant activity. FVIII recovery and half‐life thus obtained showed that this treatment was more effective than a FVIII infusion performed by way of comparison. These results indicate that this vWf defect may account for FVIII deficiency in patients without the usual laboratory and clinical features of von Willebrand's disease. Changes in therapy and genetic counselling following the new diagnosis in this family emphasize the need to search for such a vWf defect in patients in whom FVIII deficiency is not obviously X‐linked.
Measurement of fibrinogen binding to platelets in whole blood by flow cytometry: a micromethod for the detection of platelet activation
Authors: Warkentin Theodore E. , Powling M. J. , Hardisty R. M.
Published: March 2008

Platelet fibrinogen binding in whole blood has been measured in vitro by flow cytometry using a commercially available, fluorescein isothiocyanate (FITC)‐conjugated polyclonal antifibrinogen antibody. Fibrinogen–antifibrinogen immune complexes were formed in experimental conditions approaching antigen–antibody equivalence, but optimal reaction conditions in which their formation was prevented or minimized could be achieved. Immune complex formation was associated with fibrinogen binding to unstimulated platelets but did not significantly affect ADP‐induced fibrinogen binding. Half‐maximal fibrinogen binding occurred at about 0·4 μm ADP, and ADP‐induced fibrinogen binding continued progressively during 20 min incubation with 10 μm ADP. Fibrinogen binding correlated closely with platelet glycoprotein IIb–IIIa expression in members of a family with Glanzmann's thrombasthenia, and, in double labelling experiments, with the binding of PAC1, a monoclonal antibody that binds to GP lib–IIIa only after the exposure of fibrinogen receptors. These studies show that platelet fibrinogen binding can be reliably measured in whole blood by means of a polyclonal antifibrinogen antibody which does not discriminate between plasma and platelet‐bound fibrinogen, despite the presence of an approximately 100‐fold excess of the former.
Effects of recombinant human erythropoietin on haemolytic anaemia in mice
Authors: Cynshi Osamu , Shimonaka Yasushi , Higuchi Masato , Imai Nobuo , Suzuki Hiroshi , Togashi Mamoru , Okamoto Michio T. , Hirashima Kunitake
Published: March 2008

The effects of repeated administration of recombinant human erythropoietin (rHuEPO) were investigated in mice with haemolytic anaemia. Mice with haemolytic anaemia induced by phenylhydrazine (PHZ mice) were examined as an acute model and New Zealand black mice (NZB mice) at 13 months of age were examined as a chronic model. The plasma erythropoietin (EPO) level in PHZ mice was high and showed a strong inverse correlation with the Hb in the anaemia development period. However, it was relatively low in the recovery period from anaemia. On the other hand, the plasma EPO level in NZB mice showed a simple inverse correlation with the Hb. The rHuEPO was injected every day for a week into these mice. While a high plasma EPO level was maintained in PHZ mice, no significant effect was observed by injection with rHuEPO at dose of 600 IU/kg. However, in the recovery period from anaemia, RBC and haemoglobin in PHZ mice were increased by the rHuEPO treatment and recovered more quickly to their normal levels. In NZB mice, RBC and haemoglobin were also increased by treatment with rHuEPO at dose of 600 IU/kg. Anti‐RBC autoantibodies and anti‐EPO antibodies did not increase, while RBC and plasma EPO levels were increased by the rHuEPO treatment. These results suggest that some types of haemolytic anaemia are not always combined with high endogenous EPO levels and that exogenous rHuEPO may be effective for use in the treatment of haemolytic anaemia.
The effect of hypoxia on capillary endothelial cell function: modulation of barrier and coagulant function
Authors: Ogawa Satoshi , Shreeniwas Revati , Brett Jerold , Clauss Matthias , Furie Martha , Stern David M.
Published: March 2008

Summary. As the cells forming the luminal vascular surface, endothelium is exposed to alterations in the vascular microenvironment, such as hypoxaemia. In this report we demonstrate that hypoxia, with pO2 as low as 12–14 mmHg, was not toxic to endothelium, but reversibly modulated central cellular functions essential for maintenance of homeostasis: permeability of monolayers to solutes increased in a dosedependent manner, and cell surface coagulant properties were shifted to promote activation of coagulation. The anticoagulant cofactor thrombomodulin was suppressed and an apparently novel activator of factor X, distinct from the classical extrinsic and intrinsic systems, was induced. The hypoxia‐induced factor X activator was cell surface‐associated, had properties of a cysteine protease, had Mr corresponding to ∼ 100 kDa. based on sodium dodecyl sulphatepolyacrylamide electrophoresis (SDS‐PAGE), and isoelectric point (pI) ∼ 5.0. These findings indicate that hypoxia dynamically modulates endothelial function providing insights into the contribution of microvascular endothelial dysfunction in the pathogenesis of vascular lesions.
Susceptibility to HIV infection and AIDS in Italian haemophiliacs is HLA associated
Authors: Fabio G. , Smeraldi R. Scorza , Gringeri A. , Marchini M. , Bonara P. , Mannucci P. M.
Published: March 2008

Summary. We compared the frequencies of HLA antigens in two matched groups of 31 HIV‐seronegative and 31 HIV‐seropositive haemophiliacs, exposed during the years 1981–85 to comparable amounts and batches of presumably infectious clotting factor concentrates. The frequency of A2 was significantly higher in HIV‐seropositive than in seronegative haemophiliacs, with a relative risk (RR) of seroconversion of 3–92, whereas both Bw52 and DR4 were negatively associated with it. We also studied the distribution of HLA antigens in a larger group of 76 HIV‐seropositive haemophiliacs, who were at different clinical stages of HIV infection (CDC classes II‐IV) but were comparable for age and time elapsed since seroconversion. DR3 and DQw2 antigens were, particularly when concomitantly present, associated with a high risk of developing symptomatic HIV infection (RR = 11.79 and 25.33). Our data suggest that the HLA region controls susceptibility to infection with HIV and its progression to symptomatic disease in Italian haemophiliacs.
Parasite uptake of desferroxamine: a prerequisite for antimalarial activity
Authors: Scott Mark D. , Ranz Allen , Kuypers Frans A. , Lubin Bertram H. , Meshnick Steven R.
Published: March 2008

Summary. Desferroxamine has been shown to exhibit potent antimalarial activity. However, it is unclear as to whether desferroxamine functions by the chelation of extracellular, intra‐erythrocytic, or parasite‐associated iron. In order to determine desferroxamine's site of action, we have employed a large molecular weight dextran derivative of desferroxamine (70 kDa) and a reversible osmotic lysis technique by which erythrocytes were intracellularly loaded with this chelator. The desferroxamine‐dextran derivative has virtually identical iron‐binding characteristics to desferroxamine but, unlike desferroxamine, it is unable to cross the erythrocyte membrane. As previously shown, desferroxamine added to culture media exhibited potent antimalarial activity (mean effective inhibitory dose (ED50) ∼ 6 μm). However, extracellular desferroxamine‐dextran showed anti‐malarial activity only at very high doses (ED50≥180 μm), indicating that extracellular iron chelation is not involved in the antimalarial activity of desferroxamine. The intra‐erythrocytic entrapment of the desferroxamine‐dextran derivative also had no significant effect, except at very high concentrations, demonstrating that desferroxamine does not remove a non‐haem iron source necessary for malarial replication. The results of this study clearly suggests that the antimalarial activity of desferroxamine is directly related to its ability to enter the parasitic compartment and not due to the chelation of extra‐ or intra‐erythrocytic iron pools necessary for malarial growth.
Detection of Epstein‐Barr virus DNA by polymerase chain reaction in lymph node biopsies from patients with angioimmunoblastic lymphadenopathy
Authors: Knecht Hans , Sahli Roland , Shaw Phil , Meyer Christine , Bachmann Edith , Odermatt Bernhard F. , Bachmann Fedor
Published: March 2008

Summary. Epstein‐Barr virus DNA was detected by the polymerase chain reaction (PCR) in five lymph node biopsies from eight patients with diagnosis of angioimmunoblastic lymphadenopathy (AILD). Three pairs of specific primers detected EBV DNA sequences near the 5’end (Bam W region), the middle (BMRF 1 region) and the 3’end (Eco RI D region) of the viral genome with equal accuracy when 1 μg of DNA and 30 amplification cycles were used. When only 100 ng of DNA were screened with the BMRF 1 set of primers, a specific amplification product was still identifiable. The 593 base pair amplification product obtained using the Eco RI D set of primers was shown to contain an expected Sma I site at position 215, confirming the viral origin of sequence. Our findings indicate that lymph nodes of AILD patients frequently harbour the entire EBV genome at a high percentage of at least 1 viral copy per 15 000 human cells.
The role of alternative splicing patterns of BCR/ABL transcripts in the generation of the blast crisis of chronic myeloid leukaemia
Authors: Morgan G. J. , Hernandez A. , Chan L. C. , Hughes T. , Martiat P. , Wiedemann L. M.
Published: March 2008

Summary. Three major types of mRNA can be expressed as a result of the Philadelphia translocation, dependent on the position of the break within the BCR gene on chromosome 22. In addition. alternative splicing of the mRNA transcribed from the BCR/ABL fusion gene has been reported and it has been suggested that this may play a role in the generation of the acute phase of Philadelphia positive chronic myeloid leukaemia (CML). We have examined the fusion RNA present in 24 cases of chronic phase CML and 21 cases of patients with CML in blast crisis using the polymerase chain reaction. In no case was it possible to detect the presence of the ela2 junction which encodes the p190 hybrid protein product. We conclude that the acquisition of the p190 does not play a significant role in the generation of the blast crisis of CML. Neither could we detect a significant difference in the number of cases which simultaneously express both b2a2 and b3a2 junction products in samples isolated from chronic phase and blast crisis. In the series analysed by ethidium bromide stained gels, there was, however, an increase in the percentage of cases expressing the b3a2 junction in the mononuclear cells of blast crisis patients as compared to the white blood cells of patients in chronic phase.
ABL proteins in Philadelphia‐positive acute leukaemias and chronic myelogenous leukaemia blast crises
Authors: Ponzetto Carola , Guerrasio Angelo , Rosso Claudia , Avanzi Giancarlo , Tassinari Angela , Zaccaria Alfonso , LoCoco Francesco , Foa Robin , Basso Giuseppe , Abate Maria Lorena , Comoglio Paolo M. , Saglio Giuseppe
Published: March 2008

Summary. The Philadelphia chromosome (Ph1) is present in 95% of chronic myelogenous leukaemias (CML) and 15% of acute lymphoblastic leukaemias (ALL). This cytogenetic marker is due to a t(9;22) translocation, which causes a rearrangement of the ABL oncogene. In order to better define the relationship between type of genomic rearrangement, variant ABL protein expressed and haematological phenotype, a series of Phl‐positive acute leukaemias, both myeloblastic (AML) and lymphoblastic, and several CML lymphoid blast crises have been analysed at the DNA and protein level.

The results confirm the presence of the ABL protein P210 in all cases of CML, ALL and AML positive for rearrangement in the bcr region of chromosome 22, and, surprisingly, in one AML case apparently negative for bcr rearrangement. The ABL protein P190 was found to be present only in cases of ALL negative for bcr rearrangement. Polymerase chain reaction (PCR) analysis of the types of 9/22 junctions present in the mRNA of CML lymphoid blast crises showed no evidence of‘ALL‐type’transcripts.
Antithrombotic potencies of heparins in relation to their antifactor Xa and antithrombin activities: an experimental study in two models of thrombosis in the rabbit
Authors: Amar J. , Caranobe C. , Sie P. , Boneu B.
Published: March 2008

Summary. Our purpose was to determine the relative contribution of the antifactor Xa and antithrombin activities of heparin to its antithrombotic potency. The antithrombotic activities of unfractionated heparin (UH), two low molecular weight heparins (LMWH, CY 216 and CY 222) with increasing anti‐factor Xa/antithrombin ratio and a synthetic pentasaccharide (PS) with high affinity to antithrombin III and no antithrombin activity were evaluated. In the Wessler‐thromboplastin model, the most potent antithrombotic agent, on a weight basis, was UH followed by CY 216. CY 222 and the PS which was 40 times less potent than UH. On an antithrombin unit basis, the antithrombotic potencies of UH, CY 216 and of CY 222 were equivalent. Thus, in this model, the antithrombotic effect results from the catalytic action of UH or LMWH on thrombin inhibition. In the Wessler‐serum model, on a weight basis, the antithrombotic effectiveness of UH was unchanged, those of CY 216 and CY 222 were doubled, and that of the PS was increased 10 times. On an anti‐factor Xa unit basis, CY 216 was as effective as UH, and PS as effective as CY 222. On an antithrombin unit basis, CY 216 and CY 222 were equivalent and more potent than UH. Thus, in this model, the antifactor Xa activity of heparin becomes important for its antithrombotic property.

After a single subcutaneous injection of 1000 antifactor Xa U/kg, the antithrombotic effects of UH were maintained for more than 14 h in the two models. After injection of the same dose of CY 216 significant antithrombotic effects were observed only for 9 h, in the Wessler‐thromboplastin model but for 18 h in the Wessler‐serum model. At that time, no detectable antithrombin activity was measurable in the plasma while 0.11 units of antifactor Xa activity/ml was detected. Thus, the relative contribution of the anti‐factor Xa and antithrombin activities to the antithrombotic effect of a LMWH differs according to the nature of the thrombogenic stimulus.
Reactivity of a human monoclonal antibody against Rh D with the intermediate filament protein vimentin
Authors: Thorpe Susan J.
Published: March 2008

Summary. The human IgM anti‐Rh D antibody MAD‐2 has previously been shown to react with human and animal tissues and leucocytes. Double labelling immunofluorescence with MAD‐2 and a mouse monoclonal antibody against vimentin, the intermediate filament protein of cells of mesen‐chymal origin, showed coincidental staining which was distinct from that seen with antibodies against other cytoskeletal proteins. Using immunoblotting, both MAD‐2 and anti‐vimentin reacted with a 55 kDa tissue component, and with purified vimentin. These results show that the major tissue and leucocyte protein recognized by MAD‐2 is vimentin.