bluuebunny/crossref_metadata_2025 huggingface dataset:
Cell surface expression and functional significance of adhesion molecules on human myeloma‐derived cell lines
Authors: Kim Ivana , Uchiyama Hiroshi , Chauhan Dharminder , Anderson Kenneth C.
Published: March 2008

Summary. Multiple myeloma is characterized by the presence of malignant plasma cells predominantly localized in bone marrow. Our prior studies have suggested that human myeloma derived‐cell lines adhere specifically to fibronectin and to bone marrow stromal cells (BMSCs) via β1 and β2 integrins as well as RGD peptide, and that tumour cell to BMSC contact triggers interleukin‐6 (IL‐6) secretion from BMSCs. Since IL‐6 is a growth factor for myeloma, adhesion may be important in paracrine IL‐6 mediated tumour cell growth. We therefore examined phenotypic expression of adhesion molecules on the U266 and IM‐9 human myeloma‐derived cell lines using the panel of monoclonal antibodies (MoAbs) directed at adhesion molecules submitted to the Vth International Conference on Human Leukocyte Differentiation Antigens. U266 and IM‐9 myeloma cell lines express mainly CD29, CD49d, VLA‐1, CD18, CD54, ICAM‐2 and ICAM‐3. In contrast, CD49b, VLA‐3, CD49f, CD11b, VCAM‐1, selectins and selectin‐ligands were not expressed on these cell lines. Specific adherence of IM‐9 cells to BMSC line LP101 was demonstrated which could be partially blocked by pre‐incubation and culture of tumour cells with anti‐β1 integrin, anti‐β2 integrin, anti‐CD49d, anti‐VLA‐5, anti‐CD11a, anti‐CD44 and anti‐CD54 MoAbs. The combination of these MoAbs (anti‐CD29, CD18, CD11a, CD49d, VLA‐5, CD44, CD54, ICAM‐2, ICAM‐3 MoAbs) decreased but did not completely abrogate binding of IM‐9 to BMSCs. Moreover, increases in IL‐6 secretion from BMSCs after adherence of IM‐9 cells were also partially blocked by these MoAbs. These findings suggest that multiple adhesion pathways may mediate adherence of myeloma cell lines to BMSCs, localizing tumour cells in the marrow microenvironment and triggering IL‐6 secretion by BMSCs which may augment tumour cell growth.
Peripheral T‐cell non‐Hodgkin's lymphomas of low malignancy: prospective study of 25 patients with pleomorphic small cell lymphoma, lymphoepitheloid cell (Lennert's) lymphoma and T‐zone lymphoma
Authors: Siegert Wolfgang , Nerl Christoph , Engelhard Marianne , Brittinger Günter , Tiemann Markus , Parwaresch Reza , Heinz Renate , Huhn Dieter
Published: March 2008

Peripheral T‐cell lymphomas comprise a heterogenous group of low‐ and high‐grade malignancies differing in their histopathological appearance and also in clinical and prognostic aspects. We prospectively studied 25 patients with low‐grade peripheral T‐cell lymphomas: pleomorphic, small cell lymphoma (PSC) (n = 9), lymphoepitheloid (Lennert's) lymphoma (LEL) (n= 12) and T‐zone lymphoma (TZL) (n= 4). The median patient age was 55 years (range 19‐75 years); the male to female ratio was 1.5. 13 patients (52%) had limited stages (I + II), 12 patients (48%) had advanced disease (stage III + IV). 21 patients received the COPBLAM/IMVP‐16 regimen. Two patients received more intensive treatments; two received less intensive therapy. Complete remissions were achieved in 16/25 patients (64%). The median observation time of surviving patients was 30 months (range 5‐72 months). The actuarial overall survival and event‐free survival at 2 years of 21 patients receiving COPBLAM/IMVP‐16 were 69% and 35%, respectively. Intensive chemotherapy led to complete remissions in about 60% of the patients and to long‐term disease‐free survival for one‐third. The observed clinical courses illustrate the aggressive nature of PSC, LEL and TZL.
Improvement of mitomycin C‐ and cyclophosphamide‐induced thrombocytopenia and leucocytopenia by prior treatment with deoxyspergualin in dogs
Authors: Nemoto Kyuichi , Ogino Miwa , Sugawara Yumi , Abe Fuminori , Ito Junpei , Takeuchi Tomio
Published: March 2008

Deoxyspergualin (DGS) is a new immunosuppressant which has shown inhibitory haemopoietic activity. We investigated the myeloprotective effect of DSG against the haemopoietic injury of mitomycin C (MMC) or cyclophosphamide (CYC) by measuring peripheral blood cell numbers in dogs. DSG given at 5 mg/kg on days 3, 2 and 1 before, or at 10 mg/kg on either days 2 and 1 before or on day 1 before and the day of injection of MMC at 0.25 mg/kg ameliorated both the thrombocytopenia and leucopenia caused by MMC. This ameliorative effect was more evident on platelet counts than on white blood cell counts. In addition, the leucopenia and thrombocytopenia induced by a single injection of CYC at 10 mg/kg was also ameliorated by prior DSG administration. These findings suggest that DSG may be useful in protecting against the haemopoietic damage induced by chemotherapeutic agents in the treatment of cancer.
G‐CSF‐mobilized peripheral blood progenitor cells for allogeneic transplantation: safety, kinetics of mobilization, and composition of the graft
Authors: Dreger Peter , Haferlach Torsten , Eckstein Volker , Jacobs Sophia , Suttorp Meinolf , Löuffler Helmut , And Wolfgang Müller‐Ruchholtz , Schmitz Norbert
Published: March 2008

Allogeneic transplantation of peripheral blood progenitor cells (PBPC) markes the general anaesthesia of the donor unnecessary and may result in more rapid engraftment and faster recovery of the immune system. We have studied G‐CSF‐mediated PBPC mobilization in healthy donors and analysed the cellular composition of the resulting PBPC grafts. PBPC grafts were obtained from nine healthy donors (18‐67 years old) for allogeneic or syngeneic transplantation. Six donors received 10 μg/kg G‐CSF per day, the others 5‐6 μg/kg. Mobilization and harvesting were well tolerated except for moderate bone pain which occurred in all donors primed with 10 μg/kg. With 10 μg/kg, a 31‐fold (9‐62) enrichment of circulating CD34+ cells was observed with peak values constantly occurring on day 5 after the start of G‐CSF administration. Starting harvest on day 5, one to three collections on consecutive days yielded 5.5 x 106/kg (0.9‐10.7) CD34+ cells, 219 x 106/kg (106–314) T cells, and 34 x 106/kg (23–67) NK cells per 10 litres leukapheresis volume. Altogether, PBPC grafts contained 3 times more CD34+ cells, 7 times more T cells, and 20 times more NK cells than five allogeneic marrow grafts that were analysed for comparison. The yield of CD34+ cells per 10 litres apheresis volume as well as the height of the CD34+ peak in peripheral blood were inversely correlated to the age of the donor. In the donors primed with 5–6μg/kg G‐CSF the increase of circulating CD34+ cells (4–7‐fold enrichment) and the CD34+ cell yield per 10 litres leukapheresis volume (1 x 106/kg [0.8‐2‐2]) was much smaller compared with the 10μg/kg group. In conclusion, sufficient amounts of PBPC capable of restoring haemopoiesis in allogeneic recipients can be mobilized safely by administration of G‐CSF (10 μg/kg s.c. for 5 d) in healthy donors, and harvested with one or two leukapheresis procedures. Whether the large numbers of T‐cells and NK cells that are contained in the collection products may influence graft‐versus‐host and graft‐versus‐leukaemia reactivities of PBPC grafts remains to be determined.
Red cell lipid abnormalities in acquired acanthocytosis are extended to platelets
Authors: Ulibarrena Carlos , And Ana Vecino , Cesar Jesüas M.
Published: March 2008

Acquired acanthocytosis (AA) is an uncommon disease characterized by the presence of abnormal red cells (acanthocytes) in the blood smears of affected subjects. Acanthocyte membrane is enriched in cholesterol by an abnormal plasma lipoprotein. We studied the existence of similar changes in platelets of one patient with AA. Red cell cholesterol/phospholipid (Ch/PL) ratio in the patient was 1.6 (normal 1.1 ± 0.1). Phosphatidylcholine (PC) comprised 36% of total phospholipid (30.7 ± 1.8% in controls). Platelets showed aberrant morphology in the blood smears, and the ratio Ch/PL was high in comparison with normal platelets (1.4v 0.6 ± 0.1). PC comprised 52% of total PL (39.6 ± 1.9% in normal platelets). Normal platelets incubated with autologous plasma for 24 h maintained a Ch/PL ratio of 0.7, whereas this value changed to 1.4 when these cells were incubated with plasma from the patient. These results suggest that platelets of patients affected by AA acquire the same biochemical abnormality as red cells.
Anti‐GMP140 (CD62) autoantibody in a patient with autoimmune thrombocytopenic purpura
Authors: Bierling Philippe , Bettaieb Ali , Fromont Patricia , And Marina Favrin , Duedari Najib
Published: March 2008

The presence of platelet GMP140 (CD62) antibodies was analysed by the MAIPA test in 57 sera from patients with AITP and on platelets from 54 patients with thrombocytopenia of suspected immune origin. A CD62 antibody was found in only one serum. Its specificity was confirmed by an ELISA and a radioimmunoprecipitation procedure using total intact platelets and immuno‐purified GMP140. An increased amount of platelet‐associated (PA) IgG, due to in vivo fixation of GMP140 and GpIIb/IIIa antibodies, was also found on the patient platelet membrane, suggesting that GMP140 autoantibody may contribute to immune platelet destruction. No increase in PAGMP140 antibody was found on the other 54 platelet suspensions.
Subpopulations of CD34‐positive haemopoietic progenitors in fetal blood
Authors: Thilaganathan Baskaran , Nicolaides Kypros H. , Morgan Gareth
Published: March 2008

Flow cytometry was used to determine the percentage and number of circulating CD34+ cells in fetal blood from 100 pregnancies at 13‐38 weeks gestation. When expressed as a percentage of the total number of lymphocytes, the proportion of CD34+ cells decreased exponentially from a mean of 11.1% (9.2 x 107/1) at 13 weeks to 1.0% (3.0 x 107/1) at 38 weeks (r = 0.751, P < 0.0001. The use of primitive fetal blood CD34+ progenitor cells for prenatal somatic gene therapy may have distinct advantages over postnatal somatic gene therapy.
Significant amount of hepatocyte growth factor detected in blood and bone marrow plasma of leukaemia patients
Authors: Nakamura Shuji , Gohda Eiichi , Matsuo Yoshinobu , Yamamoto Itaru , Minowada Jun
Published: March 2008

Hepatocyte growth factor (HGF) has been known as a versatile functional molecule, and as being involved in the colony formation of haemopoietic progenitor cells. Clinically, an elevated HGF level in the blood has been associated with liver diseases such as fulminant hepatic failure and acute hepatitis. We have found a high level of HGF in blood and bone marrow plasma from patients with various types of leukaemia and lymphoma. In particular, 21/31 acute myeloblastic leukaemia (AML) patients showed a significant level of HGF (< 0.40ng/ml) in their blood or bone marrow plasma. The mean value of HGF in the plasma of AML patients was 2.03 ng/ml, which was higher than that in the serum of patients with acute hepatitis. This demonstrates, for the first time, evidence of frequent association of increased levels of HGF in non‐lymphocytic leukaemias, though its significance in the disease remains unknown.
A new type of pseudothrombocytopenia: EDTA‐mediated agglutination of platelets bearing Fab fragments of a chimaeric antibody
Authors: Christopoulos C. G. , Machin S. J.
Published: March 2008

In vitro agglutination of platelets leading to low automated platelet counts was observed in EDTA‐anticoagulated blood from human volunteers receiving infusions of Fab fragments of a chimaeric monoclonal antibody to platelet glycoprotein IIb‐IIIa. This pseudothrombocytopenia depended on the presence of chimaeric Fab on the platelet surface and was not seen when sodium citrate was used as anticoagulent. Preliminary evidence suggests that this phenomenon might be mediated by immunoglobulin G reactive with the human component of the chimaeric Fab. It is important to exclude pseudothrombocytopenia when low automated platelet counts are reported in association with the administration of chimaeric anti‐platelet antibodies.
Book Review
Authors: Reid C. D. L.
Published: March 2008

Book Review in this articles:

The Hemopoietic Microenvironment: The Functional and Structural Basis of Blood Cell Development. Edited by M. W. LONG and M. S. WICHA
Effects of interleukin‐6 on mobilization of primitive haemopoietic cells into the circulation
Authors: Pettengell Ruth , Luft Thomas , de Wynter Erika , Coutinho Lucia , Young Rosemary , Fitzsimmons Lesley , Scarffe J. Howard , Testa Nydia G.
Published: March 2008

Summary. Twenty‐seven patients with advanced adenocar‐cinoma were studied. Groups of three patients received interleukin‐6 (IL‐6) in doses ranging from 0.5 to 20 μg/kg by daily subcutaneous injection on days 1–7 and 22–49. Four patients received IL–6 2.5 μg/kg/d with GM‐CSF 5 μg/kg/d and three patients received IL‐6 2.5 μg/kg/d. with IL‐3 5 μg/kg/d. Circulating platelet numbers increased 1.65‐fold during IL‐6 treatment, in a dose‐dependent fashion (P= 0.01). This increase is inferior to that expected from laboratory studies. No significant change in total WBC was seen after IL‐6 alone.

After treatment with IL‐6, significant increases in numbers of circulating mononuclear cells (2.2‐fold, P= 0.006) and GM‐CFC numbers (3.2‐fold, P= 0.01) were seen, but there were no changes in circulating megakaryocyts‐CFC numbers. In contrast, after treatment with IL‐6 and GM‐CSF, larger increases in both circulating GM‐CFC (20‐fold, P= 0.04) and megakaryocyte‐CFC numbers (18‐fold, P= 0.03) were seen. Increases in blood progenitors after treatment with IL‐6 and IL‐3 did not achieve statistical significance. The ability of peripheral blood mononuclear cells to generate and sustain long‐term haemopoiesis in vitro was similar in IL‐6‐treated patients to that in untreated control subjects. No significant changes in the incidence of bone marrow progenitors or their cycling status (assessed by thymidine suicide) were seen.

These data suggest that IL‐6 alone will not be clinically useful to mobilize blood progenitor cells in cancer patients.
Diagnostic value of serum IL‐6 level in monoclonal gammopathies
Authors: DuVillard Laurence , Guiguet Michel , Casasnovas Rene‐Olivier , Caillot Denis , Monnier‐Zeller Veronique , Bernard Agnes , Guy Henri , Solary Eric
Published: March 2008

Summary. The serum level of IL‐6 was reported to reflect disease severity in patients with multiple myeloma. We used a specific radioimmunoassay to measure the level of IL‐6 in 239 serum samples in which a monoclonal gammopathy was identified for the first time. The same sample was used for the measurement of serum C reactive protein and serum albumin. Then, an inventory of clinical and biological features allowed us to classify these patients into five groups: monoclonal gammopathy of undetermined significance (MGUS: 128), multiple myeloma (MM:66), Waldenström's macroglobulinaemia (WM:27), non‐Hodgkin's lymphoma (NHL: 11) and chronic lymphocytic leukaemia (CLL: 7). The number of patients with serum IL‐6 (S‐IL‐6) level >0.335 ng/ml (upper limit in normal sera) was significantly higher in the MM group (35%; Confidence Interval (CI) 23.5–46.5) compared with the MGUS group (15%; CI 8.8–21.2). The distribution of S‐IL‐6 levels was also significantly different between the groups (Mann‐Whitney test: P< 0.01). High S‐IL‐6 levels were measured in 5/11 patients with NHL and 9/27 patients with WM. The distribution of S‐IL‐6 levels in these groups was the same as that in MGUS or MM groups. In patients with MM, elevated S‐IL‐6 levels were associated with haemoglobin level <100 g/l (P< 0.005). bone marrow plasmocytosis >50% (P<0.05) and stages II and III in the Durie & Salmon staging system (P< 0.005). The S‐IL‐6 level was also related to light chain component excretion in urine (P<0.01) and M component serum level for IgA (P<0.01). In patients with MGUS, the S‐IL‐6 level correlated with serum CRP level (P<0.05). serum lactate dehydrogenase (P< 0.05) and serum ferritin (P < 0.01). We conclude that the S‐IL‐6 level is a marker of high tumour burden in multiple myeloma. However, S‐IL‐6 level can be increased in patients with MGUS in relation to inflammatory parameters. Therefore the S‐IL‐6 level does not demonstrate high predictive value for the diagnosis of MM in patients with newly identified monoclonal gammopathy.
Immunomodulation treatment for childhood virus‐associated haemophagocytic lymphohistiocytosis
Authors: Chen Rong‐Long , Lin Kai‐Hsin , Lin Dong‐Tsamn , Su Ih‐Jen , Huang Li‐Min , Lee Ping‐Ing , Hseih Kue‐Hsiung , Lin Kuo‐Sin , Lee Chin‐Yun
Published: March 2008

Summary. The Epstein‐Barr virus (EBV), or human herpesvirus‐6 (HHV‐6) associated haemophagocytic lymphohistiocytosis, has been found prevalent in Taiwan; it affects previously healthy children and is always fatal when treated only supportively. Recognition of the underlying pathogenesis for this disease prompted adoption of an immunomodulatory regimen of intravenous immunoglobulin (IVIG) and/or etoposide on 17 such patients treated between 1990 and 1993. Remarkable improvement in patients’ prognoses was demonstrated. Eight patients are still alive with a median follow‐up of 1 year and 2 months post‐treatment. Both IVIG and etoposide had positive immunomodulation effects such as alleviation of fever and normalization of haematological and hepatic parameters. Sustained complete response was obtained in two of nine cases of EBV‐associated diseases treated with IVIG only. EBV transcripts became undetectable after etoposide and/or IVIG treatment without antiviral agents. Etoposide given by split‐dose schedule appeared to be superior to conventional three‐consecutive‐days schedule for both remission induction and disease‐free survival. Our preliminary trial apparently provides a promising improvement in the treatment of this previously fatal disease. IVIG or etoposide is effective in reversing the process of lympho‐histiocytic dysregulation resulting from virus infection of immune cells in this syndrome and probably helps hosts to control active virus replication in certain cases, through immunomodulation.
The vascular cell adhesion molecule (VCAM‐1) is expressed on a subset of lymphoid and myeloid leukaemias
Authors: Reuss‐Borst M. A. , Ning Y. , Klein G. , Muuller C. A.
Published: March 2008

Summary. The expression of the vascular cell adhesion molecule (VCAM‐1), recently identified as cytokine‐inducible ligand of the β1‐integrin VLA‐4, was investigated on normal and malignant haemopoietic precursors as well as on haemopoietic cell lines. VCAM‐1 was demonstrated on >20% blasts in 4/22 acute myeloid leukaemia (AML) and 6/10 acute lymphoblastic leukaemia (ALL) specimens but was absent from CD34+ normal bone marrow precursor cells. Interestingly, the VCAM‐1+ AMLs classified as M1 and M5 simultaneously expressed N‐CAM (CD56), a member of the immunoglobulin family. In ALL, VCAM‐1 expression was restricted to Calla+ CD19+ precursors of the c‐ALL subtype. VCAM‐1 was also found on some cell lines, mainly of the B‐lymphocytic type. Furthermore, in 13/20 chronic lymphocytic leukaemia (CLL) samples >20% of the CD19+ B‐lymphocytic precursors carried VCAM‐1, which seemed to correlate with more advanced disease. Therefore VCAM‐1 expression appeared to characterize leukaemic cells of the B‐cell lineage as well as a CD56+ subset of AML. Since its expression was clinically correlated with dermal infiltrates of leukaemic cells in AML and with advanced Rai stages in CLL, VCAM‐1 may play a role in enhanced adhesion of the malignant cells to tissues and/or to each other.
Low‐dose recombinant interleukin‐2 therapy in advanced multiple myeloma
Authors: Peest Dietrich , Leo Regine , Bloche Sabine , Hein Reinhard , Stannat‐Kiessling Sabine , Tschechne Barbara , Fett Werner , Harms Peter , Hoffmann Lutz , Bartl Reiner , Wacker Hans‐Heinrich , Gourg Siegfried , Atzpodien Jens , Kirchner Hartmut , Deicher Helmuth
Published: March 2008

Summary. In vitro data have demonstrated autologous T‐lymphocytes with anti‐tumour activity in multiple myeloma (MM). Therefore a phase I/II trial was conducted to study the feasibility, the effect on several immunological parameters, and the tumour response induction of low‐dose recombinant interleukin‐2 (rIL‐2) in MM patients. 18 MM patients of advanced stages in progress, who had failed on standard chemotherapy received 9 × 106IU/m2 rIL‐2 twice daily on days 1 and 2 and 0.9 × 106IU/m2 twice daily for 5 subsequent days per week subcutaneously from days 3 to 56 (repeated every 12 weeks until progression). Patients were treated for between 8 and 1086+d (mean 241 d) without serious side‐effects. 6/17 patients experienced tumour response (2/17 objective tumour mass reduction, 4/17 long‐lasting stable disease following tumour progression before initiation of rIL‐2 treatment). During therapy the number of eosinophils increased 15‐fold, CD4+ T lymphocytes were activated as demonstrated by enhanced CD25 antigen expression, and CD56+ NK cells expanded in the peripheral blood. Furthermore, a diminished pre‐treatment ratio of CD4+/CD8+ lymphocytes was normalized during rIL‐2 treatment. NK cell activity and lymphokine activated killer (LAK) cell activity was significantly enhanced. Endogenous IL‐2 production and elevated soluble IL‐2 receptor serum concentrations were induced. Low‐dose rIL‐2 can stimulate immune enhancement in MM despite the characteristic tumour‐induced immunodeficiency. The treatment has proven though limited efficacy in advanced MM. Because most of the responders experienced termination of tumour progression rather than tumour regression. rIL‐2 maintenance of chemotherapy‐induced remissions should be investigated.
Expression of mdrl, mrp, topoisomerase IIα/β, and cyclin A in primary or relapsed states of acute lymphoblastic leukaemias
Authors: Beck James , Handgretinger Rupert , Dopfer Roland , Klingebiel Thomas , Niethammer Dietrich , Gekeler Volker
Published: March 2008

Summary. In a series of 60 ALL samples drawn during different stages of the disease we used a cDNA‐PCR approach to analyse the relative mRNA levels of the MDR‐associated genes encoding mdrl/P‐glycoprotein, mrp, and the topoisomerase II isozymes α and β. Expression analysis of the cyclin A gene was included to examine cellular proliferation activity. The expression of gapdh served as an internal standard. Calculating the mean values we found: (i) a distinctly lower mdrl gene expression in primary ALL and first relapses compared to bone marrow from healthy donors, (ii) no change in mdrl and mrp, but a decreased topoisomerase IIα gene expression in first relapses of ALL compared to the primary leukaemia, and (iii) increased mdrl and mrp levels combined to decreased topoisomerase IIα levels in recurrent relapses of ALL showing significant correlations (mdrl/mrp: rs=+0.6833, P<0.05: mdrl/topollα: rs− 0.6727, P < 0.05). The expression of the topoisomerase IIá gene was correlated to that of cyclin A, indicating a link of its expression to cellular proliferation. Our findings suggest that a multifactorial MDR including mrp appears particularly in recurrent relapses of ALL. which often do not respond to chemotherapy. Nonetheless, some individual samples showed gene expression levels very different from the mean values calculated for a particular state of the leukaemia, indicating the need of an individual expression analysis of MDR‐associated genes.
Effects of interleukin‐12 on natural killer cell cytotoxicity and the production of interferon‐7 and tumour necrosis factor‐a in patients with myelodysplastic syndromes
Authors: OGATA K. , TAMDRA H. , YOKOSE N. , AN E. , DAN K. , HAMAGUCHI H. , SAKAMAKI H. , ONOZAWA Y. , CLARK S. C. , NOMURA T.
Published: March 2008

The effects of Interleukin 12 (IL‐12) on natural killer (NK) cell cytotoxicity and on the production of interferon‐7 (IFN‐7) and tumour necrosis factor‐a (TNF‐a) were examined in 15 patients with myelodysplastic syndromes (MDS), which are well known to have immunologic defects, and in 11 normal subjects. The NK cell cytotoxicity of all of the normal subjects was augmented by incubation with IL‐12 alone, and co‐incubation with interleukin 2 (IL‐2) further augmented it (type A response). The MDS patients showed varied responses to IL‐12/IL‐2. Seven patients showed the type A response, resulting in augmented NK cell cytotoxicity which was similar to that in the normal subjects. In five other patients the cytotoxicity was not increased by IL‐12 alone, but the combination of IL‐12 and IL‐2 did augment the cytotoxicity (type B response). The augmented cytotoxicity in these type B patients was lower than that in the normal subjects. In the final three MDS patients the cytotoxicity was low and not affected by IL‐ 12 and/or IL‐2 (type C response). AH patients with refractory anaemia with excess blasts (RAEB) and patients with RAEB in transformation showed a type B or C response. Conversely, six of eight refractory anaemia patients showed a type A response. In MDS patients there was a positive correlation between the percentage of CD3CD56+ cells in pre‐incubated cells and the cytotoxicity of cells incubated with IL‐12/IL‐2. The combination of IL‐12 and IL‐2 augmented IFN‐7 and TNF‐Q production by nonadherent mononuclear cells in a synergistic or cumulative manner, respectively, in most patients. These results suggest that IL‐12, alone or with IL‐2, may modulate these important immunologic functions in most MDS patients.
Administration of rHuGM‐CSF activates monocyte reactive oxygen species secretion and adhesion molecule expression in vivo in patients following high‐dose chemotherapy
Authors: WILLIAMS MARC A. , KELSEY STEPHEN M. , COLLINS PETER W. , GUTTERIDGE CHARLES N. , NEWLAND ADRIAN C.
Published: March 2008

Microbicidal and cytocidal products of the respiratory burst and integrin adhesion molecule expression have been studied in monocytes from patients who received rHuGM‐CSF during regeneration after high‐dose chemotherapy. In this study, administration of rHuGM‐CSF after high‐dose chemotherapy significantly augmented the secretion of inducible products of the monocyte respiratory burst. Monocyte activation persisted for several weeks after the cessation of GM‐CSF therapy. Under in vitro conditions that mimicked gram‐negative (LPS) and gram‐positive (opsonized Staphylococcus aureus) sepsis, the monocyte responded to such stimulation by exhibiting an enhanced release of hydrogen peroxide at both regeneration and several weeks later (P < 0–001). Similarly, GM‐CSF administration significantly augmented the phenotypic expression of the /32‐integrin adhesion molecules and allowed the leucocyte‐specific selectin, LAM‐1, and the /J2‐integrins to respond normally to inflammatory stimulation by LPS. We further present evidence that GM‐CSF therapy restored the otherwise refractory status of monocytes to inflammatory stimulation that existed in those patients given chemotherapy alone. The restoration of monocyte responsiveness by GM‐CSF following high‐dose chemotherapy could be a potentially valuable and hitherto not described action of rHuGM‐CSF on monocyte function. We conclude that administration of GM‐CSF may have the potential for restoring as well as augmenting the anti‐microbial and anti‐tumour function of the monocyte after high‐dose chemotherapy.
Low plasma heparin cofactor II levels in thalassaemia syndromes are corrected by chronic blood transfusion
Authors: O'DRISCOLL A. , MACKIE I. J. , PORTER J. B. , MACHIN S. J.
Published: March 2008

Low plasma heparin cofactor II (HCII) levels are associated with a thrombotic tendency, and we have previously shown these to be decreased in a variety of haemolytic conditions. The risk of thrombosis is recognized to be increased in both thalassaemia major (TM) and intermedia (TI), although the exact mechanisms are poorly understood.

HCII levels have therefore been compared in 20 untrans‐fused patients with TI and 20 regularly transfused TM patients to determine the influence of transfusion on HCII. Additionally, untransfused TI patients have been commenced on regular red cell transfusion and the effects on correction of low HCII levels investigated. HCII levels were significantly lower in the untransfused TI patients (mean 0–56 – 0–06U/ml) compared to TM patients (mean 0–85 – 0‐lU/ml; P< O'OOl). Levels in TI were significantly less than in healthy age‐matched controls (P < 0–001) and correlated with Hb values (r = 0′8), whereas levels in TM

were at the lower end of the normal range. ATIII values were within the normal reference range in both TI and TM, and HCII antigen showed a parallel reduction to HCII activity, indicating that reduction in HCII is not a consequence of increased thrombin consumption. Three patients with TI were commenced prospectively on hypertransfusion programmes which resulted in a slow normalization of their levels taking 2–3 months.

These findings support a hypothesis that the low HCII levels are related to increased red cell turnover and can be normalized once this turnover has been suppressed by hypertransfusion, The thrombotic risk to patients with low HCII levels in the presence of haemolysis might in principle be decreased by such transfusion regimes.
Idiotypic oligonucleotide probes to detect myeloma cells by mRNA in situ hybridization
Authors: BROWN Ross , LUO XIAO‐FENG , GIBSON JOHN , MORLEY ALEC , SYKES PAM , BRISCO MICHAEL , JOSHUA DOUG
Published: March 2008

An mRNA in situ hybridization (ISH) method which used non‐radioactive idiotypic oligonucleotide probes has been used to detect malignant cells in the bone marrow and peripheral blood of patients with multiple myeloma. For each of two patients with multiple myeloma a pair of biotinylated antisense oligonucleotide probes (18–22mer) was prepared from non‐germline sequences of the rearranged immunoglobulin heavy chain (IgH) gene. These oligonucleotide sequences were not homologous with any previously published sequence. The probes from each patient were specific as shown by a failure to hybridize to any cells from six other myeloma patients and four normal individuals. Specific staining of IgH gene mRNA occurred only when the myeloma cells and the sequence of the probe used were from the same patient. Using simultaneous fluorescent immunocytochemistry it was shown that more than 95% of the ISH‐positive cells expressed the malignant light chain in their cytoplasm. ISH positive cells were found in 1–4% of the peripheral blood mononuclear fraction of these two patients. These studies show that idiotypic oligonucleotide IgH gene probes can be used to identify individual cells belonging to the malignant clone and offer the possibility of developing innovative tumour‐specific therapeutic procedures using antisense technology for patients with myeloma.
Hemizygous expression of the wild‐type p53 allele may confer a selective growth advantage before complete inactivation of the p53 gene in the progression of chronic myelogenous leukaemia
Authors: NAKAI HIROYUKI , MISAWA SHINICHI , HORIIKE SHIGEO , MAEKAWA TAIRA , KASHIMA KEI , ISHIZAKI KANJI
Published: March 2008

We analysed the expression of the p53 gene in various clinical phases of chronic myelogenous leukaemia (CML) by quantitative reverse transcriptase (RT) polymerase chain reaction (PCR). Hemizygous expression of the wild‐type p53 allele was observed in two samples showing the loss of one p53 allele, and affected p53 expression was associated with p53 allelic loss rather than the clinical phase of CML. In four patients with CML showing p53 inactivation, we performed a detailed sequential analysis of p53 allelic loss, p53 mutation and expression from the onset of the disease to the patients’death. Consequently, we demonstrated that the loss of a wild‐type p53 allele preceded mutation of the remaining allele, and that cells hemizygous for the wild‐type p53 allele dominated those with both wild‐type alleles, then were replaced by cells with complete p53 inactivation. These observations indicate that not only complete p53 inactivation but also hemizygous expression of the wild‐type p53 allele may confer a selective growth advantage, and that the former is implicated in a more malignant phase than the latter. Alternatively, the inactivation of another undefined anti‐oncogene on chromosome 17p may allow selective growth before the p53 mutation occurs.
Evidence for a paracrine pathway of B‐cell stimulation in hairy cell leukaemia
Authors: SCHMID MATHIAS , SCHREZENMEIER HUBERT , STAIB G. , PORZSOLT FRANZ
Published: March 2008

It is a well‐known phenomenon that the growth of malignant B‐lymphocytes, i.e. hairy cells, is regulated by cytokines. Several investigators have suggested that the stimulating cytokines are produced by the malignant B cells themselves, indicating an autocrine growth regulation. In this paper we demonstrate that T‐lymphocyte clones produce soluble mediators which stimulate the growth of malignant B lymphocytes. The incidence of the growth‐stimulating T‐cell clones derived from peripheral blood is identical in patients with hairy cell leukaemia (HCL) and healthy controls. About 50% of the clones stimulate the growth of hairy cells, but not the growth of purified B lymphocytes of healthy donors. The stimulating activity of a single clone varies when tested on different hairy cells. Interferon alpha (IFNa), but not antibodies against tumour necrosis factor alpha (TNFQ) or interleukin‐2 (IL‐2), completely inhibit the growth‐stimulating activity. Our results indicate that a paracrine growth regulation has to be considered in addition to the postulated autocrine loop in the growth regulation of malignant B cells.
A low Mr GTP‐binding protein, Rapl, in human platelets: localization, translocation and phosphorylation by cyclic AMP‐dependent protein kinase
Authors: ATA KOH‐ICHI NAG , NOZAWA YOSHINORI
Published: March 2008

Subcellular fractions were prepared from human platelet membranes by sucrose density gradient centrifuga‐tlon and the localization of a low Mr GTP‐binding protein, rapl protein (Rapl) was analysed by immunoblotting using a specific antibody. Rapl, which has been purified from human platelets, was found to be located in plasma membrane and a‐granule fractions in resting platelets. Treatment of isolated a‐granules with pronase led to proteolysis of Rapl, indicating that this protein is exposed to the cytoplasmic face of the granules. Degranulation of a‐ granules consists of translocation and subsequent fusion of the granules with the open canalicular system. Activation of this process by thrombin induced the redistribution of Rapl on the a‐granules to plasma membranes. On the other hand, Rapl is known to be phosphorylated by cyclic AMP‐dependent protein kinase (A‐kinase) in vitro and in vivo. In intact human platelets, phosphorylation of Rapl by A‐kinase in response to prostaglandin Ex (PGEi) was observed only in Rapl localized in plasma membranes and not on a‐granules, although Rapl was phosphorylated in a cell‐free system when plasma membranes and a‐granule membranes were exposed to A‐kinase as substrates. These results strongly suggest that Rapl in plasma membranes and the protein on a‐granules are regulated by different mechanisms, and have different functions.
Refractory cytopenia with t(l;7), + 8 abnormality and dysplastic eosinophils showing intranuclear Charcot‐Leyden crystals: a fluorescence in situ hybridization study
Authors: MA S. K. , WONG K. F. , CHAN J. K. C. , KWONG Y. L.
Published: March 2008

A case of refractory cytopenia and marrow eosinophilia showing t(l;7) translocation and concomitant trisomy 8 is reported. The eosinophils were dysplastic, and showed the unique feature of intranuclear Charcot‐Leyden crystal formation, giving rise to a‘lip‐like’appearance. We speculate that this unusual cytologic feature resulted from abnormal precipitation of Charcot‐Leyden crystal protein in the eosinophils. By fluorescence in situ hybridization using a chromosome 8 specific a‐satellite probe, the abnormal eosinophils were shown to have derived from the abnormal clone. We postulate that the dysplastic clone might have retained a differentiation potential and be responsive to normal haemopoietic stimuli.
Haemoglobin Dhofar is linked to the codon 29 C –→ T (IVS‐1 nt – 3) splice mutation which causes /3+ thalassaemia
Authors: WILLIAMSON D. , BROWN K. P. , LANGDOWN J. V. , BAGLIN T. P.
Published: March 2008

Investigations of a young man with apparent thalassaemia minor showed that he was a heterozygote for a rare abnormal haemoglobin variant, Hb Dhofar. The amino acid replacement is in the /3‐globin chain (j358 Pro – Arg) and is therefore not consistent with the observed proportion of Hb Dhofar, as in both this and the original case, it constituted only 15% of the total haemoglobin. We have determined the basis of the low expression of this mutant, which is due to its linkage to a thalassaemic splicing mutation on the same /3‐globin gene at codon 29 (C –> T). The finding of this thalassaemia mutation linked to Hb Dhofar not only explains the low level of Hb Dhofar, but also provides evidence that the codon 29 C–> T, IVS‐1 splice junction mutation causes a (3+ form of thalassaemia.
Flow cytometric analysis of homologous restriction factor 20KD (HRF20) expression on progeny cells during differentiation from haemopoietic progenitors in paroxysmal nocturnal haemoglobinuria
Authors: NISHIMURA TOMOKO , KANAMARO AKIHISA , KAKISHITA EIZO , NAGAI KIYOYASU
Published: March 2008

Summary. Paroxysmal nocturnal haemoglobinuria (PNH) is an acquired clonal disorder with a deficiency of glycosyl‐phosphatidylinositol (GPI) anchored proteins. Homologous restriction factor 20KD (HRF20, CD59) is a GPI‐anchored and major complement‐regulatory protein which plays a key role in the haemolytic mechanism of PNH. We examined the differentiation stage at which the PNH abnormality occurs, by means of flow cytometric analysis of HRF20 expression. Non‐phagocytic mononuclear marrow cells were labelled with anti‐HRF20 monoclonal antibody and sorted into either HRF20‐negative or ‐positive fractions. The sorted cells were cultured in methylcellulose and their progeny in the colonies or bursts were analysed for HRF20 expression. All colonies and bursts from HRF20‐negative fractions remained negative, whereas those from HRF20‐positive fractions were either positive or negative. The possibility of a sorting error was excluded, because the secondary colonies from the HRF20 positive primary colonies consisted of both positive and negative progeny. These results suggest that there are several stages during differentiation from early progenitors to mature cells, at which the PNH abnormality becomes manifest.
Molecular analysis of the chromosomal breakpoint and fusion transcripts in the acute lymphoblastic SEM cell line with chromosomal translocation t(4;ll)
Authors: MARSCHALEK R. , GREIL J. , LOCHNER K. , NILSON I. , SIEGLER G. , ZWECKBRONNER I. , BECK J. D. , FEY G. H.
Published: March 2008

Summary. The chromosomal breakpoint and fusion transcripts of the pre‐B‐Ieukaemia‐derived SEM cell line carrying a reciprocal t(4;ll)(q21;q23) translocation were analysed. The breakpoint from derivative chromosome der4 was cloned and sequenced. The crossover site was localized in intron 7 of the ALL‐1 gene on chromosome llq23 and in a large intron of the AF‐4 (FEL) gene. RNA transcripts from both wild‐type genes and both hybrid genes were detected by reverse transcriptase polymerase chain reaction (RT‐PCR) assays. In addition, alternatively spliced mRNA species derived from the der4 chromosome were found. They were generated by using the exon 5’of the breakpoint on der4 as a common splice donor site and the 5’boundaries of exons 8 or 9 of the ALL‐1 gene as alternative splice acceptor sites. The hypothesis is proposed that selective pressure operators to maintain the presence of both derivative chromosomes as important elements in the leukaemogenic process.
Direct evidence for dissociated megakaryocytic chimaerism in a Wiskott‐Aldrich patient successfully allografted
Authors: BRUNEL VERONIQUE , MOZZICONACCI MARIE JOELLE , SAINTY DANIELLE , FAUCHER CATHERINE , ARNOULET CHRISTINE , SIMONETTI JACQUELINE , MARANINCHI DOMINIQUE , BLAISE DIDIER , LAFAGE‐POCHITALOFF MARINA
Published: March 2008

Summary. We report a Wiskott‐Aldrich patient who underwent allogeneic bone marrow transplantation from his HLA‐identical sister at the age of 25. Conditioning regimen consisted of cyclophosphamide (180mg/kg) and thoracoabdominal irradiation (6 Grays). Cytogenetic follow‐up revealed rapid and complete lymphoid chimaerism, but prolonged mixed bone marrow chimaerism. Correlative interphase cytogenetics performed on bone marrow smears using dual‐colour fluorescence in situ hybridization with × and Y specific probes showed that the proportion of donor cells was significantly higher within megakaryocytes than in other lineages. This patient therefore presented with dissociated lineage engraftment, which is not exceptional in congenital diseases and aplastic anaemia, but has not previously been described in Wiskott‐Aldrich syndrome. Bone marrow transplantation was successful despite this delayed engraftment which ensured adequate production in the involved cell lines.
T‐cell‐rich B‐cell lymphoma and lymphocyte‐predominant Hodgkin's disease: two closely related entities?
Authors: SCHMIDT U. , METZ K. A. , LBDER L.‐D.
Published: March 2008

Summary. T‐cell‐rich B‐cell lymphoma (TCRBCL) is a recently described variant of non‐Hodgkin's lymphoma. It may arise de novo or secondary to follicular lymphoma and large B‐cell lymphoma. We present here seven cases of TCRBCL to emphasize a peculiar relationship to lymphocyte‐predominant Hodgkin's disease. Morphologically, the neoplastic populations of all TCRBCLs, in addition to centro‐blast‐like and immunoblast‐like cells, comprised a few L+H‐like elements. These neoplastic cells were all regularly scattered in a majority of reactive small T‐lymphocytes as well as histiocytes. Moreover, tumour cells of TCRBCL, including the L+H‐like elements of TCRBCL, expressed LCA and L26 but did not stain for Leu‐Ml and BerH2, as is the case with the Reed‐Stemberg cell L+H variant of lymphocyte‐predominant Hodgkin's disease. Furthermore, the L26 immunoreaction in one of the cases, which otherwise presented as typical TCRBCL, disclosed a small subcapsular area resembling nodular paragranuloma because some few foci consisting of mature B lymphocytes with occasional L+H‐like elements were seen. This also holds true for a second of the TCRBCLs presented that obviously coexisted with recurrent Hodgkin's paragranuloma 10 years after the primary manifestation. These findings indicate a close connection between TCRBCL and lymphocyte‐rich Hodgkin's disease, and it may even be speculated as to whether TCRBCL represents merely a phenotypically different manifestation of this Hodgkin's subtype. Although the data presented here will not provide sufficient proof of this hypothesis, it seems clear that the nosology of TCRBCL in the context of current lymphoma classifications requires further elucidation.
Atypical chronic lymphocytic leukaemia witht(ll;14)(ql3;q32): karyotype evolution and prolymphocytic transformation
Authors: CIINEO ANTONIO , BALBONI MASSIMO , PIVA NADIA , RIGOLIN GIAN MATTEO , ROBERTI MARIA GRAZIA , MEJAK CRISTINA , MORETTI SABRINA , BIGONI RENATO , BALSAMO ROSA , CAVAZZINI PIERLUIGI , CASTOLDI GIANLUIGI
Published: March 2008

Summary. In order to define better the cytological and clinical features of atypical B‐cell chronic lymphocytic leukaemia (B‐CLL) with t(ll;14)(ql3;q32), sequential morphologic immunological and cytogenetic studies were performed in seven patients belonging to a series of 72 consecutive cases presenting with a diagnosis of CLL or atypical CLL according to the FAB criteria.

Cytologic diagnosis in these seven patients with t(ll;14) was typical CLL in two cases presenting with < 10% large lymphocytes (LL) and prolymphocytes (PL) and atypical CLL in five cases in which LL and PL comprised between 10% and 55%. The diagnosis was supported by histologic findings on bone marrow biopsy (five cases) or splenectomy specimens (two cases). A progressive increase of peripheral LL and PL was observed, resulting in a switch of FAB diagnosis over a 6‐60‐month period from typical CLL into atypical CLL in two cases and from atypical CLL into prolymphocytic leukaemia in five cases.

Immunophenotyping showed a mature B‐cell phenotype with CD19, CD22, CD24 positivity and CD10 negativity in all patients. A bright‐staining pattern for surface immunoglobulins (SIg) was detected in 6/7 cases, CD5 positivity in 6/7 cases, and CD23 positivity in 1/7 cases. The FMC‐7 monoclonal antibody was positive in >40% cells in 5/6 cases.

Chromosome changes in addition to t(11; 14) were seen in five cases; in two cases unbalanced translocations involving the 3q21 chromosome region, resulting in partial trisomy for the long arm of chromosome 3, were detected early in the course of the disease. Karyotype evolution that was associated with disease progression occurred in 3/6 assessable patients.

Comparison of these findings with similar data from 65 B‐CLL patients without t(ll;14) showed that atypical morphology, switch of FAB diagnosis during the course of the disease, and karyotype evolution were more frequently seen in cases with t(ll;14) (5/7 v 15/65 cases, P = (V015, 7/7 v 7/65 cases, P < 0‐0001, and 3/6 v 5/45 assessable cases, P= 0‐04, respectively). The frequency of positivity for CD2 3 and bright SIg staining differed significantly in the two groups.

It is concluded that t(ll;14) identifies a cytologically atypical subset of B‐CLL, characterized by frequent cytologic and cytogenetic evolution and by a distinct immunological profile, sharing some biological features with mantle cell lymphoma.
Karyotypic evolution in a granulocytic sarcoma developing in a myeloproliferative disorder with a novel (3;4) translocation
Authors: MYINT H. , CHACKO J. , MOULD S. , ROSS F. , OSCIER D. G.
Published: March 2008

Summary. A 59‐year‐old man presented with a granulocytic sarcoma arising in the manubrium, and splenomegaly. The blood count showed 1‐2 × 109/1 eosinophils and a marrow aspirate was hypercellular with eosinophilia. Cytogenetic analysis of the marrow revealed a novel t(3;4) (pl3;ql2) and analysis of cells aspirated from the granulocytic sarcoma showed the same abnormality and an additional trisomy 8.

Intensive chemotherapy and local radiotherapy led to resolution of the chest mass but persistence of the chromosome translocation in the marrow.
Flow cytometric analysis of intracellular CD6 8 molecule expression in normal and malignant haemopoiesis
Authors: STROBL HERBERT , SCHEINECKER CLEMENS , CSMARITS BETTINA , MAJDIC OTTO , KNAPP WALTER
Published: March 2008

Summary. CD68 molecules are heavily glycosylated lysosomal membrane constituents of unknown function with strong expression in monocytes and macrophages. Using flow cytometry, we quantified expression levels of CD68 molecules in normal and malignant haemopoietic cells. CD68 molecules are intensely expressed in the cytoplasm and weakly on the surface of mature CD14+ monocytes. CD68 expression seems to start very early during granulo‐monopoietic differentiation. Virtually all myeloperoxidase (MPO)+ bone marrow cells coexpress CD68 and similar proportions of CD34+ progenitor cells weakly express CD68 or MPO molecules. During further differentiation, CD68 expression is strongly up‐regulated in early MP0+ precursor cells which lack lactoferrin (LF) and CD14 molecules. Compared to these, more mature MPO+LF+ bone marrow and peripheral blood granulocytes express considerable lower levels of CD68. In‐line with this broad expression, all investigated acute myeloid leukaemia (AML) cases, classified as FAB M1‐M5, were CD68 positive, and compared to normal CD34+ bone marrow cells, CD34+ AML blast cells expressed increased levels. CD68 expression is, however, not restricted to cells of myeloid origin, because a subset (40 – 15%, n = 6) of CD19+ peripheral blood B‐lympho‐cytes and 50% of B‐ALL are also weakly positive. In contrast, normal CD3+ lymphocytes lack (<3%. n = 6) CD68 and only low proportions (6 – 3%, n= 6) of CD56+ NK cells are CD68+. Also, all investigated T‐ALL cases (n = 6) lacked CD68.