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Myeloproliferative disorders are associated with increased risk of thrombosis and vascular complications. The pathogenesis of these complications is not completely known. Reactive oxygen species produced by the neutrophil NADPH oxidase could have a role in this process. The aim of this study was to evaluate reactive oxygen species production by neutrophils of myeloproliferative disorder patients. Patients with or without the JAK2 V617F mutation were characterized. Reactive oxygen species production was assessed by chemiluminescence, and phosphorylation of the NADPH oxidase subunit p47phox was analyzed by Western blots. In a comparison of controls and myeloproliferative disorder patients without the JAK2 V617F mutation, reactive oxygen species production by neutrophils from patients with the JAK2 V617F mutation was dramatically increased in non-stimulated and in stimulated conditions. This increase was associated with increased phosphorylation of the p47phox on Ser345 and of the uspstream kinase ERK1/2. In neutrophils from healthy donors, JAK2 can be activated by GM-CSF. GM-CSF-induced p47phox phosphorylation and priming of reactive oxygen species production are inhibited by the selective JAK2 inhibitors AG490 and lestaurtinib (CEP-701), supporting a role for JAK2 in the upregulation of NADPH oxidase activation. These findings show an increase in reactive oxygen species production and p47phox phosphorylation in neutrophils from myeloproliferative disorder patients with the JAK2 V617F mutation, and demonstrate that JAK2 is involved in GM-CSF-induced NADPH oxidase hyperactivation. As neutrophil hyperactivation could be implicated in the thrombophilic status of patients with myeloproliferative disorders, aberrant activation of JAK2 V617F, leading to excessive neutrophil reactive oxygen species production might play a role in this setting.

V617F JAK2 mutation is a reliable molecular marker of polycythemia vera (PV), potentially useful to monitor the effect of treatments in this disease. In a phase 2 study of pegylated (peg) IFN-alpha-2a in PV, we performed prospective sequential quantitative evaluation of the percentage of mutated JAK2 allele (%V617F) by real-time polymerase chain reaction (PCR). The %V617F decreased in 24 (89%) of 27 treated patients, from a mean of 49% to a mean of 27% (mean decrease of 44%; P < .001), and no evidence for a plateau was observed. In one patient, mutant JAK2 was no longer detectable after 12 months. In 3 patients homozygous for the mutation, reappearance of 50% of wild-type allele was observed during treatment. The results seem to confirm the hypothesis that IFN-alpha preferentially targets the malignant clone in PV and show that %V617F assessment using a quantitative method may provide the first tool to monitor minimal residual disease in PV. This trial was registered at www.clinicaltrials.gov as #NCT00241241.

Fludarabine (FDR) therapy gives a response rate of about 30% in previously treated patients with Waldenström's macroglobulinemia (WM). The combination of FDR and cyclophosphamide (Cy) has been shown to be effective in chronic lymphoproliferative disorders. We administered the combination of FDR (30 mg/m2 i.v. D1-D3) and Cy (300 mg/m2 i.v. D1-D3) to 49 patients. Median age was 64 years. The median hemoglobin, albumin, beta 2 microglobulin and immunoglobulin M (IgM) levels were 9.9 g/100 ml, 39.6 g/l, 3 mg/l and 24.7 g/l, respectively. In all, 14 patients (29%) had not previously been treated. FDR/Cy was administered every 4 weeks for a median of four cycles. In all, 38 patients (77.6%) had partial responses, nine had stable disease and two had progressive disease. After a median of follow-up of 25 months, six patients relapsed and two patients developed large-cell lymphoma. The median time to treatment failure was 27 months. The main toxicity was hematological. In all, 12 patients died, four from progression, one from large-cell lymphoma, three from infection and four from a second malignancy. Two factors negatively influenced overall and event-free survival, age >65 years and IgM <40 g/l. The FDR/Cy combination, therefore, gives a high response rate in WM, even in previously treated patients with factors of poor prognosis.

We report a case of severe priapism occurring in a patient with unstable hemoglobin, hemoglobin Köln, and underline several factors that may have contributed to this complication: abnormal plasticity of red cells, splenectomy, and cytomegalovirus infection. Since emergency treatment may prevent impotence, patients and parents should be educated about this complication., (Copyright 2003 Wiley-Liss, Inc.)

The appearance of schistocytes in a peripheral blood film is considered to be an important diagnostic marker for thrombotic microangiopathy. However, the morphological analysis of schistocytes remains uneasy. To determine practice patterns in the biological management of schistocytosis, the French Group of Cellular Hematology from the French Society of Hematology conducted a survey on the approach of the diagnosis of microangiopathy. A guideline is proposed in order to cancel the substantial variation among biologists.

First described in 1948, Bernard-Soulier syndrome is an uncommon hereditary thrombopathy characterized by abnormal expression of the GPIb-IX-V complex which inhibits platelet migration to the site of endothelial trauma. Our case illustrates the pathophysiological mechanisms involved and points out the similarity with idiopathic thrombopenic purpura.

Background: Treatment of adults with autoimmune thrombocytopenic purpura (AITP) is based more on individual experience than on results of controlled studies. We compared intravenous immunoglobulin with high-dose methylprednisolone in untreated adults with severe AITP and assessed efficacy of subsequent oral steroids compared with placebo. Primary outcome was number of days with platelet count greater than 50 x 10(9)/L within the first 21 days., Methods: We did a randomised multicentre trial based on a 232 design. 122 adults with severe AITP (platelet count < or =20 x 10(9)/L) were randomly assigned to receive either intravenous immunoglobulin or high-dose methylprednisolone on days 1-3 (randomisation A), and then to receive either oral prednisone or placebo (randomisation B) on days 4-21. Analysis was by intention to treat., Findings: Six patients were excluded from the analysis. The number of days on which platelet counts were above 50 x 10(9)/L was 18 in 56 patients receiving intravenous immunoglobulin and 14 in 60 receiving high-dose methylprednisolone (p=0.02). Percentage of patients who had platelet counts over 50 x 10(9)/L on days 2 and 5 was 7% and 79%, respectively, in the intravenous immunoglobulin group compared with 2% and 60%, respectively, in the high-dose methylprednisolone group (p=0.04). During the second treatment period, prednisone was more effective than placebo for all short-term endpoints. Patients who received intravenous immunoglobulin and prednisone had platelet count greater than 50 x 10(9)/L for 18.5 days (p=0.005), and those treated with high-dose methylprednisolone and prednisone had this count for 17.5 days., Interpretation: Intravenous immunoglobulin and oral prednisone seems to be more effective than high-dose methylprednisolone and oral prednisone in adults with severe AITP, although the latter treatment is effective and well tolerated.

Spherocytic red cells with reduced membrane surface area are a feature of hereditary spherocytosis (HS) and some forms of autoimmune hemolytic anemia (AIHA). It is generally assumed that membrane loss in spherocytic red cells occurs during their sojourn in circulation. The structural basis for membrane loss in HS is improper assembly of membrane proteins, whereas in AIHA it is due to partial phagocytosis of circulating red cells by macrophages. A hypothesis was formed that these different mechanisms should lead to temporal differences in surface area loss during red cell genesis and during sojourn in circulation in these 2 spherocytic syndromes. It was proposed that cell surface loss could begin at the reticulocyte stage in HS, whereas surface area loss in AIHA involves only circulating mature red cells. The validity of this hypothesis was established by documenting differences in cellular features of reticulocytes in HS and AIHA. Using a novel technique to quantitate cell surface area, the decreased membrane surface area of both reticulocytes and mature red cells in HS compared with normal cells was documented. In contrast, in AIHA only mature red cells but not reticulocytes exhibited decreased membrane surface area. These data imply that surface area loss in HS, but not in AIHA, is already present at the circulating reticulocyte stage. These findings imply that loss of cell surface area is an early event during genesis of HS red cells and challenge the existing concepts that surface area loss in HS occurs predominantly during the sojourn of mature red cells in circulation.

Liver involvement in multiple myeloma (MM) has been reported very rarely in living patients. Here we describe a rare case in whom investigation of liver nodules by biopsy, revealed MM. The Role of cytadhesin molecules in the spread of plasma cell neoplasia is discussed and a review of the Literature is given.

The occurrence of leukemic pleural effusion is a rare complication in chronic lymphocytic leukemia and has not been reported in B-cell prolymphocytic leukemia (B-PLL). We report a case of pleural effusion revealing a B-PLL. The diagnosis was made on the cytological and immunological characteristics of cells in the blood and pleural effusion. This patient was treated with fludarabine and was in complete remission after three courses. This observation may have clinical implications for the use of new adenoside nucleotide analogues in symptomatic B-PLL.

We report the cytogenetic results obtained in 81 multiple myeloma (MM) patients with abnormal karyotypes. Most karyotypes were complex with numerical and structural abnormalities but the analysis of chromosomal abnormalities allowed identification of two cytogenetic patterns depending on the chromosome number: a first hyperdiploid pattern (54%) with recurrent trisomies 3, 5, 7, 9, 11, 15 and 19 and a second pattern (46%) showing either pseudodiploid, hypodiploid or near-tetraploid karyotypes. Structural abnormalities were present in all but five hyperdiploid karyotypes, and frequently involved lymphoid breakpoints: immunoglobulin gene regions (36 cases) or chromosome 11q13 region (21 cases). Numerous other structural aberrations were detected; the most frequent involved chromosome 1 and chromosome 13. Structural abnormalities were significantly more frequent in the second hypodiploid group. When analyzing the results obtained in the 60 patients studied at the time of diagnosis, a prognostic correlation was found between the cytogenetic pattern and overall survival: hyperdiploid patients had a longer survival than patients belonging to the pseudo/hypo/near-tetraploid group (median survival 36.8 vs 18.2 months, P < 0.04). These results suggest that MM could correspond to two closely related diseases.

We investigated reactive oxygen species (ROS) involvement in polymorphonuclear neutrophilic leukocyte (neutrophil) apoptosis triggering. Neutrophils were incubated with xanthine oxidase (XO), which produces superoxide anion (O2.-) and hydrogen peroxide (H2O2) or glucose oxidase (GO), which produces only H2O2. Both XO and GO accelerated apoptosis when compared to spontaneously aged neutrophils. Catalase inhibited both spontaneous apoptosis and XO- or GO-accelerated apoptosis, but superoxide dismutase did not. Hydrogen peroxide can enter the cell, thus generating intracellular oxidation, which was observed by flow cytometry. Furthermore, the intracellular reduced glutathione content fell in the presence of XO or GO; however, apoptosis was not accelerated in the presence of buthionine sulfoximine (BSO), suggesting that the fall in glutathione in the presence of XO or GO is a consequence of oxidative stress but not a trigger of apoptosis. Hydrogen peroxide can react with iron to form hydroxyl radicals (HO.); we observed that two iron chelators, deferoxamine and hydroxybenzyl ethylenediamine (HBED), both inhibited spontaneous and accelerated apoptosis, suggesting that HO. may mediate neutrophil apoptosis.

Aims: To compare by a prospective study in high risk polycythemia vera (PV) patients 33P alone and 32P followed by low-dose hydroxyurea (HU) maintenance therapy. Toxicity, efficiency, and leukemogenic potential were studied., Patients: 483 patients with a documented PV, aged more than 65 years at diagnosis, were included between 1980 and 1996 in a prospective study comparing 32P alone and 32P followed by low-dose HU maintenance therapy. Blood cell counts were performed every two months and a clinical evaluation by a specialist was obtained every four or six months., Results: Treatments were well tolerated, but chronic leg ulcers were observed in the maintenance therapy arm. The risk of leukemia was about 15% at the 15th year in the group of patients treated by 32P alone, but reached 30% in the group receiving maintenance therapy. In both arms, there was no significant correlation between occurrence of leukemia and the total dose of 32P. There was a correlation between the leukemic risk and disease severity, estimated on the frequency of relapse. Cancer occurrence was slightly higher than expected in the maintenance arm. HU treatment did not protect against progression to myelofibrosis, probably due to the lack of maintenance of an efficient myeloid or megakaryocytic suppression. Median life-span was slightly shorter in the group receiving HU maintenance. In all cases, life-span was only one year lower than that observed in the reference population., Conclusion: For all these reasons, we suggest the us of 32P alone in elderly patients; complementary chemotherapy should only be prescribed in the cases with short-term relapse, and late resistance to 32P.

Viral infections are thought to play a part in some cutaneous drug reactions. Human herpesvirus 6 (HHV6), which is the agent of exanthema subitum (sixth disease), has never been implicated in a drug reaction. We report a patient with severe phenobarbital-induced anticonvulsant hypersensitivity syndrome in whom a fulminant haemophagocytic syndrome was associated with HHV6 infection. We discuss the possible role of HHV6 in this reactive condition.

An analysis of the risk of progression towards leukemia, carcinoma and myelofibrosis was performed in 93 patients treated by 32P alone (PVSG protocols) since 1970-1979, 395 patients over the age of 65 years treated by 32P with or without maintenance therapy using hydroxyurea (French protocol) since 1980-1994, and 202 patients under the age of 65 treated by either hydroxyurea or pipobroman since 1980. The risk of leukemia, or myelodysplasia, or lymphoma in the 32P-treated patients was 10% at the 10th year, but increase after that time to reach a value of about 30% at the 20th year, in the surviving case. This risk was not dose-related. Despite a marked reduction of the cumulative 32P dose in the patients maintained by hydroxyurea, the actuarial risk was 19% at the 10th year. In the patients treated exclusively by non radio-mimetic agents (hydroxyurea or pipobroman) a risk of 10% at the 10th year was observed. The risk of carcinoma (excluding skin cancers) was about 15% at the 10th year in the 32P-treated cases, a value similar to that generally reported by the French statistics. There was no prevalence of digestive carcinomas. In contrast, the patients receiving 32P and hydroxyurea as maintenance had an excess risk: 29% at the 10th year. In the relatively young cases treated by non radio-mimetic agents, the risk was similar in both arms: 9% at the 10th year, similar to the expected incidence at this age. The risk of myelofibrosis with myeloid metaplasia was still relatively low at the 10th year, about 15% in all arms, but increased towards a value higher than 30% in the patients surviving at the 20th year. At the present time, but in only a few cases with long-term following, no myelo-fibrosis with splenic metaplasia has been observed in the pipobroman-treated cases. The present results, which need to be confirmed (the present analysis has been done in spring 95) suggest that:-the use of non radio-mimetic agents does not protect against leukemic transformation, which may be a consequence of the disease; rather than of the treatment,-maintenance therapy after initial use of 32P increases the risk of both leukemia and carcinoma,-and hydroxyurea does not delay the risk of developing myelo-fibrosis, in comparison with 32P alone.

Cytogenetic studies in multiple myeloma (MM) have been disappointing due to the low mitotic index of plasma cells. Recently the detection of clonal chromosomal abnormalities at diagnosis seemed to be improved by addition of cytokines (IL-6 and GM-CSF) in the culture medium. We performed two parallel total bone marrow cells culture types in 33 stage I, II and III multiple myeloma patients at diagnosis: 3 d without any cytokine, and 4-7 days stimulated with IL-6 and GM-CSF. No clonal chromosomal abnormality was detected in the 12 stage I and II patients either in 3 d or in 4-7 d culture. In stage II patients, abnormalities were observed in 18/21 (85.7%) and in 8/18 (44.4%) in the 3 d culture and the 4-7 d stimulated cultures respectively. Our results suggest that in stage III multiple myeloma at diagnosis, 3 d culture without cytokine may be the better technique to detect clonal chromosomal abnormalities, and, before using cytokines as a reference condition, this 3 d unstimulated culture should be considered.

We report a case of multiple myeloma with unusual cytological features. Cytological diagnosis was rendered difficult due to plasma cells with multilobulated and convoluted nuclei and this variant of myeloma seems to indicate poor prognosis.

Opsonized zymosan is able to induce polymorphonuclear leucocyte activation as reflected by lysosomal degranulation and light emission. However this stimulus is a weak inducer of arachidonic acid metabolism in these cells. We have studied the effects of co-incubation of polymorphonuclear leucocytes and platelets, each of them exposed with its specific stimulus (i.e. opsonized zymosan and thrombin). The granule constituent release was enhanced in the presence of activated platelets, but not the chemiluminescence. The same results were found in time-course experiments. Another important effect of this co-incubation was the potentiation of the synthesis of 5-lipoxygenase metabolites of arachidonic acid by activated platelets. The in vitro study of cellular cooperation may be of great interest in evaluating the role of the messenger processes occurring from one cell to another.

Hypercalcemia was associated with osteolytic bone lesions in a 60-year-old woman with chronic myelogenous leukemia in the accelerated phase. Using highly specific antisera to parathyroid hormone, radioimmunoassays disclosed elevated levels of carboxyl-terminal (53-84) and intermediate (44-68) fragments. In addition, concomitant variations of serum calcium level and leukocyte counts, increased urinary c-AMP excretion, morphological integrity of parathyroid glands, and absence of bone resorbing activity in myeloblast culture supernatants are consistent with the hypothesis that the humoral hypercalcemia was due to the excessive production of PTH. This production may have been ectopic, although no PTH secretion was demonstrated in myeloblast culture supernatants.

In four members of a family presenting hereditary elliptocytosis erythrocytes were studied, membranes were extracted and proteins were analysed by gel electrophoresis in a polyacrylamide gradient. In one of the patients suffering from severe haemolytic anaemia successfully treated by splenectomy, an almost complete deficiency in band 4(1) was discovered. Endogenous protein kinase activities revealed the absence of radioactivity of band 4(1) in the proband, a result not modified by cAMP. The kinase activity was normal in the parents, which confirms the almost complete absence of protein 4(1) seen in the stained gel of the proband. A moderate increase in the phosphorylation of band 3 was observed in all the members of this family. Deformability was measured by a visco-diffractometric method (Ektacytometry) in a medium of low viscosity (11 cp at 22 degrees C), allowing the recording of curves characteristic of elliptocytosis and revealing a markedly reduced deformability index (DI). At low shear stress, elliptocytes were oriented perpendicular to the flow (a result common to all elliptocytosis). In the proband several years after splenectomy, the DI was extremely reduced at high shear stress, which can be explained by the simultaneous presence of elliptocytes, schizocytes and spherocytes. A more detailed comparative study of the proteins of the various members of the family could lead to more precise information on the possible role of band 4(1) as a linkage protein maintaining the erythrocyte membrane stability.

We present haematological and immunological data obtained in 11 patients with the rare association of chronic lymphocytic leukaemia (CLL) and multiple myeloma (MM). CLL occurred before MM in six cases and both diseases were diagnosed simultaneously in five. With regard to CLL, no peculiar clinical feature was found; in six patients, MM was characterized by extra-osseous lesions. A significant remission (3-7 years) was obtained in three patients. In six patients, CLL and plasma cells synthesized Ig molecules with different light chains, probably indicating the coexistence of two distinct clonal proliferations. In two cases where monoclonal Ig synthesized by both types of cells had the same type of light chains, studies with anti-idiotypic antibodies showed that the two proliferations were also distinct. The pathophysiology of this rare association is discussed in the light of these findings and of data from the literature.

A new technique for simultaneously measuring the phagocytic and bactericidal capacity of human leukocytes is proposed. The method uses 14C labelled bacteria and is based on the principle that only viable intra-cellular bacteria incorporate 3H-thymidine. Phagocytosis is measured by the ratio intra-cellular 14C/extra and intra-cellular 14C and the bactericidal capacity of leukocytes by the difference between the 3H-thymidine incorporation of the ingested and non-ingested bacteria. Results in normal subjects and in a case of chronic granulomatous disease show the validity of the method which is easier and quicker than the methods previously used.

The presence of vacuolated polymorphonuclear neutrophils in blood smears of patients suffering from infection appears to be associated with massive bacterial growth and to constitute a very early symptom of rapidly life-threatening septicaemia. When these cells persist for more than 36 hours, the disease may be considered as beyond control.

We report the case of a patient with an ileal tumor initially diagnosed as a lymphoma. The development of acute myeloblastic leukemia 8 months later made it clear that the initial tumor was a granulocytic sarcoma (GS). This case emphasizes the possible difficulties of histologic diagnosis and the importance of the routine use of histochemical and immunologic techniques in the diagnosis of extranodal lymphomas.

Prostaglandins, thromboxanes and leukotrienes are oxygen metabolites of arachidonic acid forming a family of lipidic substances with intrinsic biological activities. The significance of biosynthesis of these mediators in response to cell stimulation remains unclear. Numerous data suggest that these compounds, produced by blood cells and vascular cells, play an important role in cardiovascular pathology, allergy or inflammation. The concept, based on biochemistry, of distinct metabolic pathways should be reappraised, using a physiological approach that involves a biological effect resulting from a synergistic action of different compounds. The development of specific assay methods will permit to investigate compounds obtained from biological fluids or from cells present in recognized pathological situations. Such in vitro data should help to clarify the role of these autacoids in cardiovascular, allergic or inflammatory diseases.

Two cases of Henoch-Schönlein purpura in adults are reported. Both patients presented with severe digestive symptoms suggesting malabsorption or exudative enteropathy. Roentgenological examinations showed marked anomalies of the small intestine as well as changes suggestive in intra-mural hematoma located in the stomach in one case and in the duodenum in the other. Hypoalbuminemia occurred in both patients during the course of the disease and was highly suggestive of protein-loosing enteropathy. Disseminated intravascular coagulation was seen in one patient. Both patients recovered from their gastrointestinal disease. Roentgenologic changes disappeared in one case; in the other, marked compression and distension of the duodenum were still visible six months after onset.

Hepatitis-associated (Australia) antigen (HAA) was detected in the sera of 16 (50%) of 33 patients with Hodgkin's disease; all of these patients had received the same treatment for approximately two years, and they had been in complete remission for at least two years. The HAA-positive patients had significantly higher levels of serum SGPT and significantly lower bromsulphalein clearance than the HAA-negative patients. Histological changes compatible with a diagnosis of chronic persistent hepatitis were found in the livers of 12 of the 16 HAA-positive patients and in five of the 17 HAA-negative patients (p < 0.05). In 128 patients with Hodgkin's disease who had received various forms of treatment and who were studied at various stages of remission, HAA was found in the sera of 42 (33%). Tests for HAA repeated four months later in positive reactors of both groups showed persisting antigenaemia. Hepatitis-associated antigen was not present in the sera of any of 36 patients with Hodgkin's disease studied when the diagnosis was first made and before treatment had begun. These observations suggest that persistence of HAA and the presence of chronic persistent hepatitis were more likely to be related to the treatment the patients had received than to the disease itself.