bluuebunny/crossref_metadata_2025 huggingface dataset:
Autoantibodies directed against the epidermal growth factor‐like domains of thrombomodulin inhibit protein C activation in vitro
Authors: Oosting Janine D. , Preissner Klaus T. , Derksen Ronald H. W. M. , Groot Philip G. de
Published: March 2008

Summary No consensus has been obtained about the question whether autoantibodies, in particular antiphospholipid antibodies (aPL), may cause thrombosis by inhibiting thrombomodulin (TM) mediated protein C activation. In order to clarify the mechanism by which autoantibodies inhibit TM‐mediated protein C activation, we have screened 12 patients with autoimmune diseases for the presence of circulating autoantibodies inhibiting TM function. In a cross‐sectional study we found that IgG fractions from two patients (who were aPL negative) inhibited TM mediated protein C activation in an assay system using purified components. A longitudinal study of six patients with a history of thrombosis of which two were aPL positive showed that all had at some time circulating antibodies inhibiting TM function, suggesting that the presence of these antibodies is transient.

Three different TMs were used to identify the epitope of the antithrombomodulin antibodies (aTM): rabbit TM, which contains the entire TM molecule: Solulin, which contains the extracellular part of TM, and rEGF‐TM, which contains the six epidermal growth factor (EGF) domains of TM. We showed that the aTM inhibited protein C activation mediated by all three TMs, indicating that the aTM are directed against the region containing the EGF domains.

When TM was incorporated in phospholipid vesicles, no inhibition by these aTM could be demonstrated. In addition, protein C activation mediated by cultured endothelial cells (EC) could not be inhibited by aTM. The lack of inhibition of TM in phospholipid vesicles and EC‐TM by aTM suggests that aTM only inhibit soluble TM.

In conclusion, we demonstrated the transient presence of circulating autoantibodies directed against the region of TM containing the EGF domains in SLE patients with a history of thrombotic complications. We postulate that the presence of antibodies to soluble TM may be, in addition to aPL, a risk factor for the occurrence of thrombosis in patients with autoimmune diseases.
Activation of monocytes for the immune clearance of red cells in β°‐thalassaernia/HbE
Authors: Wanachiwanawin W. , Siripanyaphinyo U. , Fucharoen S. , Wasi P. , Mawas F. , Wiener B. , Wickramasinghe S. N.
Published: March 2008

Summary We have recently provided evidence that IgG antibodies play a role in the destruction of red cells in thalassaemia syndromes. In order further to delineate factors involved in the clearance of thalassaemic cells, monocytes of 30 Thai patients with β°‐thal/HbE (17 non‐splenectomized and 13 splenectomized) and 16 normal controls were examined for their ability to bind and phagocytose normal red cells coated with IgG anti‐Rh(D). In β°‐thal/HbE. the mean number of red cells attached to the monocytes was approximately 3‐fold greater than in normal controls and the number ingested 30% higher. Among the non‐splenectomized patients, the number of red cells attached to and ingested by the monocytes, correlated inversely with mean basal Hb levels, suggesting that activation of mononuclear phagocytes for the immune clearance of red cells is a factor in determining the severity of the anaemia. As Fc‐gamma‐RI is of primary importance in the recognition of IgG‐coated red cells by monocytes, leucocytes from 10 β°‐thallHbE patients (four non‐splenectomized and six splenectomized) and five normal controls were investigated for their expression of Fc‐gamma‐RI by flow cytometry. In β°‐thallHbE there was an approximately 3‐fold increase in the percentage of leucocytes expressing this receptor: the receptor was up‐regulated on monocytes and induced on granulocytes. The up‐regulation of Fc‐gamma‐RI in β°‐thallHbE is likely to be an important component in the activation of monocytes and in mediating their enhanced effector function towards antibody‐coated cells.
Low‐dose high‐frequency enzyme replacement therapy for very young children with severe Gaucher disease
Authors: Zimran A. , Hadas‐Halpern I. , Zevin S. , Levy‐Lahad E. , Abrahamov A.
Published: March 2008

Summary Six children with a mean age of 4.6 years (range 2.5–7). suffering from severe Gaucher disease, were treated with low‐dose high‐frequency intravenous enzyme replacement (Ceredasea®. Genzyme. U.S.A.) for a period of 10–24 months. Although, in general, these patients were more severely affected than previously reported patients, the results of the treatment were as satisfactory as those obtained by using much higher doses at low frequency. In addition to regression of organomegaly and improvement of haematological abnormalities, we observed two unique clinical responses in three patients: two showed decreased tendency to bacterial infections, associated with improvement in neutro‐phil chemotaxis, and one patient, with type 3 Gaucher disease, showed some improvement in neurological findings. Several measures were taken to ameliorate the burden of the high‐frequency treatment. These included implantation of venous access devices, establishment of a home‐treatment programme and the application of effective local anaesthesia. Therefore the low‐dose high‐frequency protocol appears to be both an effective and feasible alternative to the costly high‐dose low‐frequency protocols even in very young children.
Distinguishing β2‐glycoprotein I dependent (systemic lupus erythematosus type) and independent (syphilis type) anticardiolipin antibody with Tween 20
Authors: Matsuda Juzo , Saitoh Noriko , Gohchi Kengo , Gotoh Moritaka , Tsukamoto Miyo
Published: March 2008

Summary We investigated whether or not the use of Tween 20 could help to distinguish β2‐glycoprotein I (GPI) independent anticardiolipin antibody (aCL) (syphilis‐type aCL) from GPI‐dependent aCL (SLE‐type aCL) in a GPI‐dependent/ independent aCL ELISA. aCL was positive in all 16 SLB patients arid all 15 syphilis patients, who were positive for aCL in the standard ELISA. in the GPI‐independent ELISA with Tween 20. GPI‐dependent aCL was detected in 12/16 SLE patients by the GPI‐dependent ELISA with Tween 20. aCL was not detected in any of the syphilis patients by GPI‐dependent ELISAs. On the basis of these results, we recommend that Tween 20 should be used in ELISAs to distinguish GPI‐dependent aCL from GPI‐independent aCL.
Consistent involvement of the 3’half part of the first BCR intron in adult Philadelphia‐positive leukaemia without M‐bcr rearrangement
Authors: Nakamura Yuichi , Hirosawa Shinsaku , Aoki Nobuo
Published: March 2008

Summary About one half of Philadelphia (Ph)‐positive acute leukaemia (AL) patients and rare cases of Ph‐positive chronic myelogenous leukaemia (CML) show rearrangements within the first intron of the BCR gene on chromosome 22. We studied breakpoints within the first BCR intron in 22 adult patients with Ph‐positive leukaemia; 21 with AL and one with CML, which lacked rearrangements within the major bcr (M‐bcr). With a series of genomic probes from this intronic region, we detected chromosomal breaks in all 22 patients within the 35 kb area, corresponding almost to the 3’half portion of the intron. The breakpoints were distributed throughout this region but we could not identify any special cluster of breakpoints in this area. Our data support consistent involvement of the 3’half part of the first BCR intron in Ph‐positive leukaemias without M‐bcr rearrangement, and indicate relatively wide scattering of breakpoints in this portion of the intron.
Analysis of multidrug‐resistance (MDR‐1) glycoprotein and CD56 expression to separate monoclonal gammopathy from multiple myeloma
Authors: Sonneveld P. , Durie B. G. M. , Lokhorst H. M. , Frutiger Y. , Schoester M. , Vela E. E.
Published: March 2008

Summary Monoclonal gammopathy of undetermined significance (MGUS) is different from multiple myeloma (MM) by a low proliferation and by its indolent clinical course. In this study, two biological parameters were investigated which mark the transition from MGUS to MM, i.e. expression of the P‐170 glycoprotein associated with the multidrug resistance phenotype (MDR‐1) and expression of the natural killer cell antigen, CD56. Strong MDR‐1 expression was found in plasma cells of 32/38 untreated MGUS as compared with 33/105 untreated MM stage I–III (84%v 32%, P<0.001) and in 0/10 normal plasma cell samples. CD56 expression in high density was present in 43/57 analysed untreated MM but in none of 23 MGUS (78%v 0%P<0.0001). Plasma cells did characteristically show a low Ki‐67 proliferation index in 14/15 MGUS patients (mean 0.05%, range 0–0.2%) and a higher index in 25 analysed MM patients (mean 2.31%, range 1–7%, P<0.03).

These data indicate that MDR‐1 expression together with absence of CD56 expression and a low proliferation index can be used to separate MGUS from MM.
Type I von Willebrand disease, subtype ‘platelet low’: decreased platelet adhesion can be explained by low synthesis of von Willebrand factor in endothelial cells
Authors: Federici Augusto B. , De Groot Philip G. , Moia Marco , Ijsseldijk M. J. W. , Sixma Jan J. , Mannucci Pier Mannuccio
Published: March 2008

Summary Endotheial cells (EC) were isolated from the umbilical vein of a newborn girl with type I ‘platelet low’ von Willebrand disease (I vWD) and endothelial localization and release and the ability of subendothelial von Willebrand factor (vWF) to support platelet adhesion were compared with those of normal EC. vWF was detectable by immunofluorescence in Weibel‐Palade bodies, but the number of Weibel‐Palade bodies positive for vWF was lower than in control EC. Patient EC released into the medium significantly smaller amount of vWF, both constitutively and after their stimulation. The vWF content of the extracellular matrix of patient EC was 38% that of control EC matrix. Platelet adhesion studies were performed under flow conditions with umbilical arteries and EC matrices of cultured EC. Using normal citrated whole blood as perfusate, platelet adhesion was lower in the umbilical artery of the patient (9 ± 1%v 35 ± 4% for the control) and in her EC matrix (7 ± 1%v 21 ± 2% of control). When patient EC matrix was perfused with vWF‐deficient reconstituted blood, adhesion was 17 ± 3%v 32 ± 3% for control EC matrix; preincubation of patient EC matrix with 1 U/ml vWF increased the adhesion to 30 ± 6%. These data establish that low contents of vWF in EC and subendothelium are important characteristics of type I vWD ‘platelet low’, and that such characteristics correlate with low platelet adhesion to the subendothelium.
Molecular basis and haematological characterization of β‐thalassaemia major in Taiwan, with a mutation of IVS‐1 3’end TAG → GAG in a Chinese patient
Authors: Chiou S. S. , Chang T. T. , Chen P. H. , Lee L. S. , Chen T. S. , Chang J. G.
Published: March 2008

Summary We studied 41 patients with β‐thalassaemia major and their parents by using a combination of polymerase chain reaction (PCR) amplification, slot‐blot hybridization of allele‐specific oligonucleotide (ASO), and direct genomic sequencing. Eight different point mutations were characterized. C to T substitution at nucleotide (nt) 654 of intervening sequences (IVS) 2, accounting for 46.3% of mutant β‐globin genes, is the most common mutation in Taiwan, followed by frameshift codons 41/42 with four nucleotides (TCTT) deletion for 31.7%, A to G substitution at position −28 of promotor area for 8.5%, A to T substitution at codon 17 for 6.1%, frameshift codons 27/28 (insertion of C) for 2.4%, G to T substitution at nucleotide 1 of IVS‐1 for 2.4%, frameshift codons 71/72 (insertion of A) and IVS‐1 3’end TAG→GAG for 1.2%. The former four mutations showed no obvious difference between two major ethnic groups in Taiwan. As to mutations in each individual of β‐thalassaemia major, the incidence of compound heterozygotes of two different mutations is much higher than homozygotes of single mutation, 78.0%v 22.0%. Compound heterozygotes of C to T substitution at nt 654 of IVS‐2 and frameshift codons 41/42 with four nucleotides deletion is the most common pattern of β‐thalassaemia mutations in each individual (41.5%). The results are somewhat different from other documented reports concerning the mutations of β‐thalassaemia in southern China. This is the first report of mutation of IVS‐1 3’end TAG→GAG which causes consensus change in Chinese people. Patients with heterozygotes of β° and –28 β+‐thalassaemia mutations would have a greater delay in initial transfusion in comparison to patients with homozygotes of both β°‐thalassaemia mutation, but their initial clinical manifestation might be aggravated when combined with a glucose‐6‐phosphate dehydrogenase (G‐6‐PD) deficiency and an insult such as exposure to infection and certain drugs.
Cytomegalovirus viraemia and specific T‐helper cell responses as predictors of disease after allogeneic marrow transplantation
Authors: Ljungman Per , Aschan Johan , Azinge Joey N. , Brandt Lena , Ehrnst Anneka , Hammarström Viera , Klaesson Sven , Linde Annika , Lönnqvist Berit , Ringdén Olle , Wahren Britta , Gahrton Gösta
Published: March 2008

Summary Risk factors for cytomegalovirus viraemia and disease, the relation between viraemia and disease, effect of antiviral treatment, and T‐helper cell response to cytomegalovirus antigen were analysed retrospectively among 279 patients who underwent bone marrow transplantation at Huddinge Hospital. Ninety‐one of 279 (32.6%) patients developed viraemia. Donor and recipient pre‐transplant serologic status and degree of acute graft‐versus‐host disease were independent risk factors for viraemia. Forty‐nine patients (17.6%) developed cytomegalovirus disease and 44 of these patients had viraemia. Seventeen patients (6%) developed cytomegalovirus pneumonia and 14 of these patients had preceding viraema. Among patients with viraemia, acute graft‐versus‐host disease and total body irradiation were risk factors for pneumonia. Antiviral treatment initiated within 7 d of development of viraemia was associated with lower risk for development of pneumonia (P < 0.05). Sixty‐seven patients with viraemia were repeatedly tested by lymphocyte stimulation with cytomegalovirus antigen. No patient who developed cytomegalovirus pneumonia had measurable specific helper T‐cell response at the time of viraemia detection compared to 42% of patients with other concurrent or subsequent cytomegalovirus disease, and 75% of patients without subsequent disease. We conclude that viraemia is a major risk factor for development of cytomegalovirus disease. Furthermore, early antiviral treatment based on detection of viraemia can be effective in preventing cytomegalovirus disease. The length of antiviral treatment might be decided through measurements of the helper T‐cell response to cytomegalovirus antigen.
Identification of a nonsense mutation at amino acid 584–Arginine of platelet glycoprotein IIb in patients with type I Glanzmann thrombasthenia
Authors: Gu Jian‐ming , Xu Wen‐feng , Wang Xiao‐dong , Wu Qing‐yu , Chi Cheng‐wu , Ruan Chang‐geng
Published: March 2008

Using Southern blot, the restriction digests of genomic DNAs in 11 patients with Glanzmann thrombasthenia from 10 unrelated kindreds were probed with a full‐length GPIIb cDNA. An addiitonal 2.3 kb Taq I fragment and two 1.65 kb and 0.65 kb fragments with reduced band intensity were found in the genes of two affected siblings from a family originating from the city of Huang Yan in the Zhejiang province. The Taq I digest of the abnormal gene was further probed with three portions of GPIIb cDNA, revealing that the heterozygous mutation was present in the region around exons 15‐17 of the GPIIb gene. Two primers for polymerase chain reaction (PCR) were then designed, and a 394 bp PCR product was generated and sequenced, indicating that a stop codon (TGA) was substituted for an Arg codon (CGA) at amino acid position 584 of GPIIb, and resulted in a premature termination of translation and production of a shortened protein. The Western blot analysis showed that GPIIIa at the platelet surface was apparently deficient, it may be ascribed to the rapid turn‐over of GPIIIa uncomplexed with the truncated GPIIb. The abnormal 2.3 kb Taq I fragment was used as a specific genetic marker to detect the carrier status of the patient family. The abnormal allele was proved to be derived from the mother, the two affected siblings are double heterozygotes, and one clinically unaffected daughter has also inherited this defective allele, while the father carries another recessive abnormal allele unidentified.
Effect of antiphospholipid antibodies on procoagulant activity of activated platelets and platelet‐derived microvesicles
Authors: Galli M. , Bevers E. M. , Comfurius P. , Barbui T. , Zwaal R. F. A.
Published: March 2008

We have recently described the in vitro mechanism of action of anticardiolipin (aCL) and lupus anticoagulant (LA) antibodies in patients with the antiphospholipid syndrome. LA antibodies inhibit coagulation reactions in plasma because they appear to recognize the complex of lipid‐bound (human) prothrombin, whereas aCL antibodies require β2‐glycoprotein I (β2‐GPI) for binding to anionic phospholipids. aCL antibodies can be divided into two subgroups, according to their behaviour in lipid‐dependent coagulation reactions: aCL‐type A enhances the anti‐coagulant effect of β2‐GPI, whereas aCL‐type B does not.

In the present study we investigated the effect of purified aCL‐type A and B and of LA antibodies on the procoagulant activity of both Ca‐ionophore activated platelets and platelet‐derived microvesicles, using an assay system with highly purified bovine coagulation factors Xa, Va, and prothrombin from human and bovine origin. In the absence of β2‐GPI neither type of aCL was able to inhibit the prothrombinase activity of platelets or microvesicles. However, a strong and dose‐dependent inhibition of the prothrombinase activity of both platelets and platelet‐derived microvesicles was observed within a few minutes, when aCL‐type A antibodies were added in combination with β2‐GPI. This inhibitory effect was dependent also on the concentration of β2‐GPI. Conversely, no inhibitory effect of aCL‐type B antibodies on platelet‐ (or microvesicle) prothrombinase activity in the presence of β2‐GPI could be observed.

LA antibodies were able to inhibit in a dose‐dependent way the procoagulant activity of activated platelets and platelet‐derived microvesicles. With two LA preparations this inhibition was only apparent when human prothrombin was used as substrate, while a third preparation exhibited its inhibitory effect both in the presence of human and bovine prothromin.

The data indicate that, in the presence of their respective cofactors β2‐GPI and prothrombin, aCL and LA antibodies interact with the membrane of activated platelets and platelet‐derived microvesicles in a very similar way as previously observed for their interaction with anionic phos‐pholipid surfaces.
Iron status in red cell pyruvate kinase deficiency: study of Italian cases
Authors: Zanella Alberto , Berzuini Alessandra , Colombo Maria Bernadette , Guffanti Anna , Lecchi Lucilla , Poli Francesca , Cappellini Maria Domenica , Barosi Giovanni
Published: March 2008

Summary. We investigated the iron status of 33 pyruvate kinase (PK) deficient patients, most of the cases reported in Italy. Serum ferritin (SF) was higher than the upper limit of the range of matched controls in 15/25 (60%) non‐transfused patients (median 228 μg/l, range 58–3160 v 43, 22–310). Liver siderosis and fibrosis were found in 8/9, and cirrhosis in two who died at age 39 and 42 of complications of iron overload.

SF was independent of age, sex, or severity of haemolysis. The prevalence of HLA‐A3 antigen in PK deficient patients was not significantly different from that of our healthy population (29.6%v 23%). The HLA‐A3 positive, non‐transfused patients had significantly higher SF values than the HLA‐A3 negative ones (median 675 μg/l, range 340–3160 v 145, 58–400). A pedigree study of six high SF‐probands indicated that iron overload has a multifactorial pathogenesis. In particular, the association of PK deficiency‐induced haemolysis, splenectomy and an additional factor (heterozygosity for idiopathic haemochromatosis, ineffective erythropoiesis) leads to severe iron accumulation. We suggest that monitoring iron status would be useful in PK deficient patients, particularly in splenectomized and HLA‐A3 positive ones, to identify those at risk of iron overload and prevent the clinical consequences of iron accumulation.
Circulating megakaryocyte progenitors in myeloproliferative disorders are hypersensitive to interleukin‐3
Authors: Kobayashi Shoko , Teramura Masanao , Hoshino Shigeru , Motoji Toshiko , Oshimi Kazuo , Mizoguchi Hideaki
Published: March 2008

Summary. Previous studies have reported that megakaryocyte progenitors in myeloproliferative disorders (MPD) formed spontaneous megakaryocyte colonies without the addition of megakaryocyte colony‐stimulating factor (Meg‐CSF). To determine whether this spontaneous colony formation is due to autocrine proliferation of MPD megakaryocyte progenitors or to hypersensitivity to Meg‐CSF that might exist in the culture system, we investigated colony‐forming unit‐megakaryocytes (CFU‐Meg) in the peripheral blood of 11 MPD patients, using serum‐free cultures. Spontaneous megakaryocyte colonies were observed in serum‐free cultures of nonadherent mononuclear cells (NAdMNC) obtained from MPD patients with thrombocytosis, whereas the NAdMNC of MPD patients without thrombocytosis, that of patients with reactive thrombocytosis and normal subjects never formed spontaneous colonies. However, the spontaneous colonies from MPD patients with thrombocytosis disappeared in cultures using highly purified CD34‐positive cells as target cells.

To study the hypersensitivity of megakaryocyte progenitors to Meg‐CSF, dose‐response experiments were performed with interleukin‐3 (IL‐3). CFU‐Meg from MPD patients with thrombocytosis showed maximal growth at the concentrations of IL‐3 lower than those for normal subjects. CFU‐Meg of MPD patients without thrombocytosis and that of patients with reactive thrombocytosis showed the same colony growth response to IL‐3 as that of normal subjects. This result indicates that the CFU‐Meg of MPD patients with thrombocytosis are hypersensitive to IL‐3. It also suggests that spontaneous colony formation by NAdMNC is not due to the autocrine growth of megakaryocyte progenitors but is due to the hypersensitivity of megakaryocyte progenitors to Meg‐CSF, such as IL‐3, released by accessory cells. Furthermore, it is possible that such hypersensitivity of CFU‐Meg to IL‐3 might be a pathogenic factor in MPD with accompanying thrombocytosis.
Possible role of macrophages in the pathogenesis of ethanol‐induced bone marrow damage
Authors: Wickramasinghe S. N. , Hasan R.
Published: March 2008

Summary. Adherent‐cell‐depleted human marrow cells (MC) were cultured on their own or co‐cultured with monolayers of blood‐monocyte‐derived macrophages or marrow‐derived adherent cells with and without 2 mg ethanol/ml for 24 h. The incorporation of 3H‐thymidine and 3H‐leucine by MC cultured on their own was not significantly influenced by ethanol. By contrast, the incorporation of both radiolabelled compounds was significantly lower in MC from the cocultures containing ethanol than from those not containing ethanol. This effect was mediated by a diffusible factor produced by macrophages in the ethanol‐containing cultures and was independent of intercellular contact. Supernatants from ethanol‐containing cultures of marrow‐derived adherent cells displayed cytotoxic activity against A9 cells due to the presence of unstable acetaldehyde‐albumin complexes. Ethanol inhibited rather than stimulated nitrite production in the MC/marrow‐derived adherent cell co‐culture system, suggesting that macrophage‐derived nitric oxide did not play a role in causing the observed ethanol‐related effects. The data indicate that, in the presence of ethanol, macrophages cause inhibition of the incorporation of 3H‐thymidine and 3H‐leucine into overlying MC, at least partly by oxidizing ethanol to acetaldehyde and releasing some of the potentially cytotoxic acetaldehyde thus formed extracellularly. Bone marrow macrophages may therefore play an important role in the pathogenesis of alcohol‐related marrow damage in vivo.
Plasma fibrinogen in women: relationships with oral contraception, the menopause and hormone replacement therapy
Authors: Lee Amanda J. , Lowe Gordon D. O. , Smith William C. S. , Tunstall‐Pedoe Hugh
Published: March 2008

Summary.Plasma fibrinogen was measured in 4837 women aged 25‐64 years as part of the Scottish Heart Health Study and Scottish MONICA population surveys. The relationships of oral contraceptive use, the menopause and hormone replacement therapy were examined.

Univariate analyses found that women with a history of oral contraceptive use, premenopausal women and those on hormone replacement therapy all had significantly lower fibrinogen levels than women who had never used oral contraceptives, postmenopausal women and non‐hormone replacement users respectively. These differences persisted after age standardization.

On multivariate analysis, menopausal status and hormone replacement therapy had independent effects on fibrinogen levels. Together with the common risk factors, 9.9% of the total variation in plasma fibrinogen levels was explained. However, less than 1% of this was from the combined menopausal and hormonal factors.

These results confirm a postmenopausal rise in fibrinogen level which may be relevant to an increased risk of coronary heart disease. In addition, a protective effect with hormone replacement therapy is noted, although this was probably due to selection bias.
Severity differences in β‐thalassaemia/haemoglobin E syndromes: implication of genetic factors
Authors: Winichagoon Pranee , Thonglairoam Varaporn , Fucharoen Suthat , Wilairat Prapon , Fukumaki Yasuyuki , Wasi Prawase
Published: March 2008

Summary. Genetic factors determining the difference in severity of anaemia in β‐thalassaemia/HbE disease were studied in 90 patients who had haemoglobin levels, at steady state, ranging from 4.2 to 12.6 g/dl. Co‐inheritance of α‐thalassaemia 2 and haemoglobin Constant Spring could significantly decrease the severity of the disease. Inheritance of a β‐thalassaemia chromosome with Xmn I cleavage site at position — 158 of the Gγ‐globin gene which was linked to the haplotype ‐ + ‐ ++ or ++ ‐ ++, was associated with a milder anaemia. Two copies of these alleles were necessary to produce a significant clinical effect. Increased expression of the Gγ‐globin gene and higher production of haemoglobin F. which could reduce the overall globin chain imbalance, were also associated with homozygosity for the Xmn I cleavage site and thus with less severe anaemia. However, this effect was not seen in Xmn I site heterozygotes. Whether the effects of the Xmn I polymorphism, HbF concentration and Gγ/Aγ ratio act separately or through common mechanisms in reducing anaemia remains to be ascertained.
A role for complement as the major opsonin in the sequestration of erythrocytes from elderly and young donors
Authors: Shapiro Sarah , Kohn David , Gershon Harriet
Published: March 2008

Summary. Erythrocytes from elderly donors (> 70 years), but not young donors (18‐35 years), are shown to undergo sequestration in an in vitro erythrophagocytosis assay. Comparable levels of sequestration are observed for high density erythrocytes from young individuals and both low density and high density erythrocytes from elderly individuals. These cells, which are susceptible to phagocytosis with no additional treatment are collectively termed ‘in situ aged’erythrocytes. We present evidence for the involvement of complement in the sequestration of ‘in situ aged’erythrocytes and correlate levels of complement bound to ‘in situ aged’erythrocytes from young and elderly donors with levels of phagocytosis. We also demonstrate that the in vitro sequestration of erythrocytes from elderly donors can be inhibited by β‐galactosyl sugars and arginine‐glycine‐aspartic acid (RGD) but not by mannose nor by Protein‐G, a specific inhibitor of Fc‐γ mediated phagocytosis. These experiments show that IgG is not the major opsonin in the sequestration of red cells from elderly donors. In support of the role of complement rather than IgG as the major opsonin in red cell sequestration, we further demonstrate that C′3 bearing immune complexes block in vitro sequestration of erythrocytes from elderly donors. This competition is not dependent upon the isotype of the immunoglobulin (IgM or IgG) in the complex but rather on the presence of active complement.
A unique case of t(15;17) acute promyelocytic leukaemia (M3) developing into acute myeloblastic leukaemia (M1) with t(7;21) at relapse
Authors: Jubashi Tohru , Nagai Kazuhiro , Miyazaki Yasushi , Nakamura Hideo , Matsuo Tatsuki , Kuriyama Kazutaka , Tomonaga Masao
Published: March 2008

Summary. This report describes a case of t(15;17) acute promyelcytic leukaemia (APL, FAB subtype M3) with dysgranulopoiesis at diagnosis in a patient who developed myelodysplasia (MDS) and then a second phenotype of t(7;21) acute myeloblastic leukaemia (AML, FAB subtype M1) at the time of relapse. To our knowledge, there is no report of a second phenotype of AML occurring after complete remission (CR) of APL. Furthermore, this is the first report of chromosomal abnormality t(7;21) in a case of AML. Several hypotheses for this unusual course of APL are discussed.
Megakaryocyte and erythroid colony formation in essential thrombocythaemia and reactive thrombocytosis: diagnostic value and correlation to complications
Authors: Juvonen Eeva , Ikkala Eero , Oksanen Kalevi , Ruutu Tapani
Published: March 2008

Megakaryocyte and erythroid colony formation in vitro by progenitors from the bone marrow and/or blood was studied in 61 patients with essential thrombocythaemia (ET) and 22 patients with reactive thrombocytosis (RT) using the methyl cellulose assay. 47 (77%) of the patients with ET showed megakaryocyte and/or erythroid spontaneous colony formation while 14 (23%) patients did not have any kind of spontaneous colonies. Spontaneous megakaryocyte colony formation was seen in 42 (69%) of the patients and 36 (59%) ET patients showed spontaneous erythroid growth. 31 patients had both types of spontaneous colonies. 11 patients showed only spontaneous megakaryocyte colony formation, and five patients only spontaneous erythroid growth. None of the patients with RT or of the normal controls showed either type of spontaneous growth. Neither the presence of spontaneous megakaryocyte colony formation nor the number of spontaneous colonies correlated with the platelet count. Patients with spontaneous megakaryocyte growth had significantly more often thromboembolic or haemorrhagic problems than those without spontaneous colony formation. In conclusion, it was found that a great majority of patients with ET but none of those with RT grow spontaneous megakaryocyte and/or erythroid colonies. Spontaneous colony formation is strong evidence for a myeloproliferative disorder. The presence of spontaneous colony growth is associated with an increased risk of thromboembolic or haemorrhagic complications regardless of the platelet count, particularly among young patients.
Carrageenan‐induced activation of human platelets is dependent on the phospholipase C Pathway
Authors: Hatmi Mohamed , Randon Jacques , Faili Ahmad , Vargaftig B. Boris
Published: March 2008

Stimulation of washed human platelets by the pro‐inflammatory polysaccharide carrageenan is accompanied by shape change, aggregation and release of granule contents and unaccompanied by thromboxane A2 synthesis. Carrageenan triggers platelet activation through a prostaglandin synthetase‐independent mechanism. The phospholipase A2 (PLA2) inhibitor, p‐bromophenacyl bromide suppresses platelet responses to carrageenan (Vargaftig et al, 1980) probably by mechanism (s) other than those which involve PLA2 activity. Exposure of platelets to carrageenan (2–25 μg/ml) induced inositol phosphate formation in a time‐ and concentration‐dependent manner, the level of inositol phosphate formation correllating with the intensity of aggregation. Neomycin, an aminoglycoside antibiotic which inhibits the phospholipase C‐mediated phosphatidylinositol 4,5‐bisphosphate breakdown, suppressed both platelet activation and inositol phosphate formation. Inhibition was concentration‐dependent with an IC50 value of about 180 μM. Platelet‐activating factor (PAF) is not responsible for carrageenan‐induced platelet activation and inositol phosphate formation, since exposure of platelets to carrageenan (25 μg/ ml) in the presence of compound WEB 2086 (100 μM). a PAF antagonist, failed to inhibit carrageenan responses. However, compound Ro 19‐3704, a structurally related antagonist of PAF reported to be also an inhibitor of phospholipases A2 and C, inhibited concentration‐dependently (0.1–10 μg/ml). These findings indicate that carrageenan activates human platelets through a phospholipase C‐dependent mechanism and show that neomycin, at low concentrations, can be a selective inhibitor of phospholipase C‐mediated PIP2‐breakdown.
Immunological effects of intermediate purity clotting factor concentrates: failure to affect lymphocyte activation in vivo
Authors: Higgins Julie A. , Giangrande Paul L. F. , Kernoff Peter B. A. , Goodall Alison H.
Published: March 2008

Concern has been expressed that intermediate purity clotting factor concentrates may cause immunological abnormalities in haemophilic patients, distinct from those related to HIV infection. Early reports of lymphocyte dysfunction in anti‐HIV seronegative haemophiliacs pointed to activation of their lymphocytes; a potential cause of CD4 + ve lymphocyte decline in anti‐HIV seropositive patients. Recent reports have suggested that the use of high purity FVIII concentrates might retard the rate of decline in CD4 + ve lymphocytes in haemophiliacs infected with the HIV virus.

Expression of markers of acute and chronic activation of T and B lymphocytes was measured in heavily treated anti‐HIV seronegative haemophiliacs using two‐colour flow cytometry. No T or B lymphocyte stimulation was observed. Cellular markers of activation were absent and CD4 + ve lymphocyte counts and serum IgG levels were normal. Anti‐HIV seropositive haemophiliacs showed T and B cell activation consistent with HIV infection. The extent of lymphocyte activation in individual patients was unrelated to the type, amount or frequency of FVIII received.

These findings do not support the hypothesis that lymphocytes of haemophiliacs are affected directly by the regular administration of intermediate purity concentrates so as to accelerate the progression of HIV disease.
The diagnostic significance of serum ferritin indices in patients with malignant and reactive histiocytosis
Authors: Lukina E. A. , Levina A. A. , Mokeeva R. A. , Tokarev Yu. N.
Published: March 2008

Iron metabolism was studied in 10 patients with malignant histiocytosis (MH), in 16 patients with histiocytosis‐X (Langerhans cell histiocytosis) and in 34 patients with reactive proliferation of the mononuclear phagocytes (MPS). Eight MH patients had a considerably increased level of serum ferritin (SF). The average level of SF was 6070 ± 957 mg/1 for MH patients, which is significantly greater than the SF level for HX and RH patients.

The study of the serum ferritin profiles suggests that the main pathogenetic mechanism of hyperferritinaemia in MH is the synthesis and secretion of ferritin by neoplastic mononuclear phagocytes.

From this, hyperferritinaemia in MH may be considered as pathognomic of this disease and used as an additional diagnostic criterion. In addition, SF is the most informative parameter for the clinical practice among the indices of the functional state of MPS.
The Progression of HIV disease in a haemophilic cohort followed for 12 years
Authors: Sabin Caroline , Phillips Andrew , Elford Jonathan , Griffiths Paul , Janossay George , Lee Christine
Published: March 2008

A cohort of haemophilic patients who seroconverted to HIV‐1 between October 1979 and July 1985 has been followed to 1 January 1992. The median age at initial seropositivity was 24 years with a range of 2–77 years. By January 1992, 38/111 (34%) had developed AIDS and 39/111 (35%) had died (four of liver failure including one hepatoma). Using Kaplan‐Meier plots, the calculated progression to AIDS at 12 years is 45% (95% CI 31, 58): for age > 25 years 63% (95% CI 45, 82), age < 25 years 32% (95% CI 15, 48) P= 0.0001; CMV positive 68% (95% CI 48, 87) CMV –ve 20% (95% CI 8, 32) P= 0.0009. The 12‐years progression rate to CD4+0.2 or AIDS is 66% (95% CI 55, 76). 21/34 (63%) of patients who are p24 antigen positive have developed AIDS compared to 17/77 (22%) who are p24 antigen negative (=0.0001). 19/34 (56%) and 20/77 (23%) of those p24 positive and negative respectively have died (P= 0.007). Before antiviral and prophylactic treatment for asymptomatic patients there were nine AIDS cases in 3.84 years experience with CD4 + <0.05 (1/0.43 years) and since treatment, 10 AIDS cases in 18.22 years (1/1.8 years). Age, CMV status and p24 remain strongly predictive of disease progression. Treatment appears to reduce the incidence of AIDS.
Danazol in autoimmune haemolytic anaemia
Authors: Pignon Jean‐Michel , Poirson Emmanuelle , Rochant Henri
Published: March 2008

Ten adult patients with warm antibody haemolytic anaemia at initial presentation and seven other patients with either refractory AiHA (two patients) or who relapsed after an initial response to prednisone (five patients) were treated with both Danazol and prednisone. 80% of the first group, but also 60% of the second group displayed long‐lasting responses (mean follow up 21 months). Minimal side‐effects occurred. Finally, addition of Danazol at presentation in warm AIHA may decrease the duration of prednisone therapy and markedly reduce the necessity of second‐line splenectomy which is usually required in many patients.
High incidence of lymphoid infiltration on labial salivary gland biopsy in non‐Hodgkin's lymphomas: clinical implications
Authors: Morel P. , Quiquandon I. , Janin A. , Dupriez B. , Desablens B. , Gosselin B. , Bauters F.
Published: March 2008

Summary. Sjögren's syndrome (SS) is characterized by an increased risk of developing a non‐Hodgkin's lymphoma (NHL). We performed labial salivary gland biopsy (LSGB) in 103 patients with untreated NHL. negative for human immunodeficiency virus. Median age was 58 years (range 21–79) and M/F 1.3. Using the Working Formulation, 37 patients had low‐grade NHL and 66 had intermediate‐ or high‐grade NHL. Dense lymphocytic infiltration (positive focus score 3 or 4) was found in 28 patients. 10 (35%) of these 28 patients fulfilled criteria for possible SS. 15/28 patients had an identical monotypic infiltrate on LSGB and NHL tissue (including two of the 10 patients with criteria for SS). The significance of the lymphoid infiltrate of LSGB was unknown in the five remaining patients with positive focus score. Significant correlations were found between positiv focus score and presence of two or more extranodal sites of disease (P=0.02), impaired performance status (P= 0.004), splenomegaly (P=0.05), increased gammaglobulin level (P= 0.03), and β2 microglobulin (P=0.004). Considering intermediate‐ or high‐grade NHL, we found significant correlation between positive focus score and unfavourable prognosis according to the two Dana Farber Cancer Institute indexes (P<0.04), to the LNH‐84 index (P=0.05), and to the international index (P=0.003). In conclusion, systematic evaluation of LSGB in 103 patients with NHL found lymphoid infiltration in 28% of them, but possible SS could be considered in only 10%. This lymphoid infiltration, though not correlated with any particular histological subtypes, was associated with unfavourable clinical prognostic factors, especially in intermediate‐ or high‐grade NHL.
Are immunoglobulins with lupus anticoagulant activity specific for phospholipids?
Authors: Pierangeli S. S. , Harris E. N. , Gharavi A. E. , Goldsmith G. , Branch D. W. , Dean W. L.
Published: March 2008

Summary. Recent studies have suggested that the lupus anticoagulant (LA) may be specific for prothrombin. prothrombin‐phospholipid complexes, or β2 glycoprotein 1 (β2, GP1) rather than phospholipids. We performed a series of experiments to determine whether LA is indeed phospholipid specific. IgG was purified from sera of six patients with the antiphospholipid syndrome (APS) and 10 healthy controls. The six IgG‐APS preparations had both LA and anticardiolipin (aCL) activity. Incubation of the six IgG‐APS preparations with cardiolipin (CL), phosphatidylserine (PS), phosphatidyl‐choline (PC), or PS/PC (20:80) liposomes in Tris‐buffered saline, resulted in loss of LA activity from the supernatant. We postulated that loss of activity might have resulted from absorption of IgG LA antibodies by phospholipids, a dilutional effect, or the presence of phospholipids in the supernatant causing ‘by‐pass’ of IgG LA inhibitory activity. To distinguish between these possibilities, we re‐isolated IgG from the supernatants and re‐tested them for LA activity. IgG re‐isolated from the PS, CL and PS/PC supernatants had no LA activity, but LA activity remained in the PC supernatant. This suggested that IgG with LA activity was absorbed by negatively charged but not by zwitterionic phospholipids. In like manner, PS, CL and PS/PC, but not PC liposomes, absorbed IgG with aCL activity. Mixtures of the phospholipid liposomes with β2GP1 did not modify the absorption of IgG with LA or aCL activity. Finally, we demonstrated that IgG eluted from immunoglobulin‐cardiolipin liposome complexes had LA activity. Based on these findings. we conclude that at least one population of antibodies with LA activity is phospholipid specific.
Sweet's syndrome associated with G‐CSF
Authors: Paydas Semra , Sahin Berksoy , Seyrek Ertugrul , Soylu Merih , Gonlusen Gulfiliz , Acar Alparslan , Tuncer Ilhan
Published: March 2008

Summary. Sweet's syndrome (SS) developed in two patients with acute myeloid leukaemia (AML) treated with granulocyte colony stimulating factor (G‐CSF) for febrile neutropenia due to AML chemotherapy. Fever, painful skin and conjunctival lesions developed and neutrophilic infiltration was detected at biopsy specimens. Neutrophilia was not detected. Skin lesions regressed within 1–2 weeks and conjunctival lesions within 4 weeks following the cessation of G‐CSF. We conclude that SS may be a complication of G‐CSF therapy and tender skin and/or conjunctival lesions developing during G‐CSF therapy should suggest the possibility of SS.
Three cases of the myelodysplastic syndrome with pericentric inversion of chromosome 16
Authors: Narayanan M. N. , Geary C. G. , Harrison C. J. , Cinkotai K. I. , Lewis M. J.
Published: March 2008

Summary. Inversion of chromosome 16, inv(l6)(pl3q22), is characteristic of acute myeloid leukaemia (AML) with eosinophilia and is rarely found in the myelodysplastic syndrome (MDS). We report three cases of MDS in which inv(16) was observed. They were classified to FAB subtypes RA. RAKS and KAEBT: eosinophilia or abnormal eosinophils were not observed. The disease appeared to be stable in all three patients. MDS with inv(16) without eosinophilia may be a rare subgroup associated with a good prognosis.
HPA‐1 typing by PCR amplification with sequence‐specific primers (PCR‐SSP): a rapid and simple technique
Authors: Metcalfe P. , Waters A. H.
Published: March 2008

Summary. A DNA‐based method was developed to genotype donors for the human platelet antigens HPA‐la and ‐1b. Sequence‐specific primers (SSP) were used in the polymerase chain reaction (PCR) which allowed the HPA‐la/la. ‐lb/lb and ‐la/lb genotypes to be determined by PCR alone, no second analytical stage was required. 10 donors were tested by PCR‐SSP and the results were concordant with serological phenotyping and independent DNA analysis.
Recombinant human erythropoietin in autologous blood donors: a dose‐finding study
Authors: Biesma D. H. , Kraaijenhagen R. J. , Dalmulder J. , Marx J. J. M. , Van De Wiel A.
Published: March 2008

A dose‐finding study of recombinant human erythropoietin (rhEPO) was performed in 60 autologous donors who donated 2 units of blood prior to orthopaedic or vascular surgery. The correction of phlebotomy‐induced anaemia was studied in four groups of 15 patients who received 500 U/kg, 250 U/kg, 125 U/kg or no (controls) rhEPO subcutaneously twice per week during a 3‐week period. Haemoglobin concentration in the 500 U/kg, 250 U/kg and 125 U/kg group and in the controls reached respectively 99·2, 98·8, 91·9 and 87·1% of pre‐phlebotomy value. Flow cytometric analysis of reticulocytes showed a steady increase of reticulocyte count as the dose increased. Maximal levels of high fluorescence reticulocytes which represent early changes of erythropoiesis were reached after 7 d and decreased thereafter in each group. Serum ferritin decreased significantly to approximately 50% of baseline values in all groups; no differences in the decrease of serum ferritin were observed between the patients who received rhEPO and the controls. No severe adverse events were observed.

This study demonstrates a dose‐related effect of rhEPO on erythropoiesis in autologous donors during the first 2 weeks. No further increase of reticulocytes was observed despite continued rhEPO therapy, which may be due to the inability of the mononuclear phagocytic system to release additional iron. To restore pre‐phlebotomy haemoglobin concentration, a dose of 250 U/kg rhEPO was sufficient.
Rapid and sensitive diagnosis of cytomegalovirus and Pneumocystis carinii pneumonia in patients with haematological neoplasia by using capillary polymerase chain reaction
Authors: Honda Junichi , Hoshino Tomoaki , Natori Hideyo , Tokisawa Shiro , Akiyoshi Hiroya , Nakahara Shin , Oizumi Kotaro
Published: March 2008

We attempted the simultaneous detection of cytomegalovirus DNA (CMV‐DNA) and Pneumocystis carinii (carinii‐DNA) in sputum samples obtained from 20 patients with haematological neoplasm with pneumonia, using rapid cycle DNA amplification (capillary PCR). We used a thermal cycler for capillary PCR which featured recirculation of hot air for rapid temperature control of 10 μl reaction samples in thin glass capillary tubes.

We extracted DNA from patients' sputa using a simple method. A comparison of the results obtained using the phenol extraction–ethanol precipitation method and those obtained using our simple method was made, and demonstrated complete agreement between the two. For detection of CMV‐DNA and P. carinii‐DNA with capillary PCR it was not necessary to vary temperature setting based on the primers used. Therefore, capillary PCR was used for the simultaneous detection of CMV and P. carinii.

After amplification, the total time required for which was 20 min, amplified products were electrophoresed on agarose gels and visualized with ethidium bromide. Product sensitivity was higher with capillary PCR than with conventional PCR.

We conclude that capillary PCR amplification is a valuable tool for rapid and simple diagnosis of CMV and P. carinii pneumonias.
Hereditary spherocytosis associated with protein band 3 defect in a Swiss kindred
Authors: Reinhart Walter H. , Wyss Erwin J. , Arnold Daniela , Ott Peter
Published: March 2008

A kindred with hereditary spherocytosis, in which 10 individuals were affected, was investigated. Gel electrophoresis of membrane proteins revealed a protein band 3 defect (densitometric reduction 14·4±7·0%). The erythrocyte morphology of unsplenectomized patients showed so‐called pincered erythrocytes (about 1%), which were not present in the five splenectomized patients. Splenectomy also reduced anisocytosis and all parameters of haemolysis, while haemoglobin increased. The osmotic resistance was reduced in patients with protein band 3 deficiency. Erythrocyte filterability through 3 μm pores was decreased. A significant correlation was found between osmotic resistance and filterability. The membrane elastic modulus of erythrocytes was not affected. These results on structural and functional properties of protein band 3 deficient erythrocytes may contribute to a better understanding of this newly discovered form of hereditary spherocytosis.