Development of inhibitors in haemophilia. Ongoing epidemiological study.

A number of 513 consecutive patients (494-haemophilia A and 19-haemophilia B) from eight haemophilia treatment centers have been investigated with Bethesda assay for the presence of factor VIII or IX inhibitors. The overall prevalence of inhibitors was 15.20%, 18.48% in severe, 5.60% in moderate and 12.24% in mild forms. The prevalence was higher than reported in most of the western countries. The age at start of substitution (p = 0.9775), the frequent switching of factor concentrates (p = 0.8931) were not relevant factors for the development of inhibitors. It is worth to be mentioned the unexpectedly occurrence of inhibitors in prior inhibitor negative (6/72) patients (during surgical interventions) probably due to their previous scarce substitution, occurrence which seems not being connected with the continuous infusion modality of factor VIII administration (p = 0.8341). In controversial situations, in the field of low titer (≤ 1 BU/ml) inhibitors for a reliable interpretation of the results the performance of recovery index and half-life time assessment of FVIII/IX was undertaken.

Exosomes as divine messengers: are they the Hermes of modern molecular oncology?

Exosomes are cell-derived vesicles that convey key elements with the potential to modulate intercellular communication. They are known to be secreted from all types of cells, and are crucial messengers that can regulate cellular processes by 'trafficking' molecules from cells of one tissue to another. The exosomal content has been shown to be broad, composed of different types of cytokines, growth factors, proteins, or nucleic acids. Besides messenger RNA (mRNA) they can also contain noncoding transcripts such as microRNAs (miRNAs), which are small endogenous cellular regulators of protein expression. In diseases such as cancer, exosomes can facilitate tumor progression by altering their vesicular content and supplying the tumor niche with molecules that favor the progression of oncogenic processes such as proliferation, invasion and metastasis, or even drug resistance. The packaging of their molecular content is known to be tissue specific, a fact that makes them interesting tools in clinical diagnostics and ideal candidates for biomarkers. In the current report, we describe the main properties of exosomes and explain their involvement in processes such as cell differentiation and cell death. Furthermore, we emphasize the need of developing patient-targeted treatments by applying the conceptualization of exosomal-derived miRNA-based therapeutics.

Cell darwinism, apoptosis, free radicals and haematological malignancies.

Haematopoiesis can be interpreted as an ecosystem composed of billions of cells interacting according to Darwinian rules. Mutation, by promoting cell diversity, ensures versatility in coping with internal and external challenges. Most mutated cells are eliminated through apoptosis. However, if mutation generates relative resistance to apoptosis it may result in growth advantage for the mutated cells. The probability of monoclonality and malignancy is significantly increased if the normal multiclonal environment is damaged by a pathologic proapoptotic process that spares the apoptosis resistant clones. Paroxysmal nocturnal haemoglobinuria, myelodysplastic syndromes, chronic myeloid leukaemia, secondary acute leukaemias and immunosuppression-related non-Hodgkin's lymphomas can be interpreted as 'opportunistic' clonal and malignant diseases. Free radicals (FRs) are closely linked to apoptosis and have been incriminated in oncogenesis. Conditions associated with increased FR formation or impaired FR disposal may provide the enhanced apoptotic background against which an apoptosis-resistant clone may gain growth advantage.

[The markers of hepatitis B, C and D viral infection in multiply transfused patients]. / Studiul markerilor infectiei cu virusurile hepatitice B, C si D la politransfuzati.

OBJECTIVE: We aimed to assess the seroprevalence of HBV, HCV and HDV virus markers in multi-transfused patients from Cluj-Napoca. MATERIAL AND METHODS: Stored serum samples of 105 multi-transfused patients (25 children, 19 adults and 61 chronically hemodialyzed patients) have been tested for HBsAg, anti-HBs, total anti-HBc, anti-HCV, total anti-HDV by automated ELISA (Sanofi Diagnostics Pasteur kits). RESULTS: HVC infection has been observed in 4/25 (16%) children, 14/19 (74%) multi-transfused adults and 48/61 (79%) haemodialysis patients. 8/25 (32%) children, 17/19 (89%) adults and 47/61 (77%) haemodialysis patients had HBV infection markers. Anti-HDV have not been found in HBV infected multi-transfused children and adults, respectively. Only 2/47 (4.25%) HBV infected haemodialysis patients had HDV infection markers. The prevalence of double infection (HCV and HBV) was high (4%, 84.2% and 67.2% in children, adults and haemodialysis patients). The prevalence of viral hepatitis markers correlated to the amount of transfused blood, and in haemodialysis patients also correlated to the duration on dialysis. CONCLUSIONS: In multi-transfused patients from Cluj area, the prevalence of viral hepatitis markers is high. The double infection (HCV and HBV) is frequent, especially in adults. The prevalence of HDV infection markers in HBV infected patients is low, in contrast with previously reported results.

Redistribution of iron towards deposits in erythroblastopenic anemia as a consequence of decreased erythroferrone production.

Most of the body iron is found within hemoglobin in red cells (the erythron), a smaller amount being distributed in other tissues such as muscles and in deposits. Iron homeostasis is a finely tuned process in which the most important regulators are probably the liver-derived hepcidin which blocks iron absorption and directs iron towards deposits and the recently discovered erythroblast-derived erythroferrone which inhibits hepcidin synthesis and therefore increases availability of iron for hemoglobin synthesis. Hepcidin secretion is increased by inflammatory cytokines and erythroferrone production increases when there is active, expanding erythropoiesis, for example after acute blood loss. We hypothesize that in pathological situations associated with erythroid precursor suppression (erythroblastopenia), anemia is the result of two major mechanisms: (1) direct erythroblast suppression leading to decreased production of red cells and (2) low iron availability due to high hepcidin levels arising as a result of low erythroferrone production. Additionally, infectious episodes and other inflammatory conditions that often complicate the course of these diseases may further promote hepcidin synthesis through increased cytokine production leading to even lower iron availability and a vicious circle of worsening anemia.

Von Willebrand factor and cardiovascular disease. Restricted relevance.

Von Willebrand's disease, a bleeding disorder, is caused by deficiencies of an endothelia-derived multimeric glycoprotein receiving the deserved name von Willebrand factor (vWf). Since this factor mediates the interaction between blood platelets and the vessels wall, it was presumed that an increase of its plasma level could be a marker of endothelial lesions and may be also involved in the pathogeny of cardiovascular disease including thrombotic complications. Actually, the unchecked release of ultralarge vWf multimers may cause widespread platelet aggregation into the microcirculation, thereby producing thrombotic thrombocytopenic purpura. More recently, it was demonstrated that such a dramatic condition is normally prevented by the intervention of a protease (ADAMTS-13) which cleaves the ultralarge and large vWf multimers into smaller multimers less active functionally although endowed with antigenic epitopes. The relevance of an immunologic assessment of vWf plasma levels as vWf:Ag should therefore be cautiously considered because this test, more accessible to clinical laboratories, does not provide information about the actual level of large multimers which are functionally active and possibly prothrombotic. It was also reported that an acute phase reaction occurring in various disease states is accompanied by increased plasma vWf levels as the result of endothelial response to adrenergic or proinflammatory stimuli. Clinical and laboratory investigations are highly suggestive that an increased plasma vWf level does not necessarily reflect its leakage from injured endothelia and the laboratory data should be considered in context with the clinical condition and the overall hemostatic balance.

Arterial stenosis and atherothrombotic events in polycythemia vera and essential thrombocythemia.

UNLABELLED: Patients with polycythemia vera (PV) and essential thrombocythemia (ET) are at risk of developing arterial and venous thromboembolic complications. Given the complex interaction between blood cells and the vessel wall, it is possible that atherogenesis may also be accelerated in these patients. We used Doppler arterial ultrasound to assess the presence of arterial stenosis in a cohort of PV and ET patients. MATERIAL AND METHODS: A total of 37 patients, 29 with PV and 8 with ET, were investigated. Aside from an extensive clinical and hematological evaluation, arterial Doppler ultrasonography was performed in all patients; in 3 patients arteriography/coronarography was also performed. RESULTS: Twenty four patients (65%) had a history of atherothrombotic events including cerebral ischemic attacks (CIA) in 12 patients, ischemic heart disease (IHD) in 10 patients and peripheral occlusive arterial disease (POAD) in 12 patients. Eight patients had multiple atherothrombotic events. Twenty five patients (67%) had other atherosclerotic risk factors such as smoking, dyslipidemia, hypertension and diabetes. Significant arterial stenosis was found in 23 patients (62.1%), including 12 patients with carotid plaques, 10 with peripheral arterial stenosis, 3 with coronary stenosis, 2 with aortic plaque and 2 with common iliac artery stenosis. In 12 patients multiple arterial stenoses were found. The presence of arterial stenosis was significantly correlated with the occurrence or thrombotic events (p = 0.0003) and was also correlated with the concomitant presence of polyglobulia and thrombocytosis. Both the thrombotic risk and the probability of stenosis detection were augmented by additional risk factors such as smoking, hypertension and dyslipidemia. DISCUSSION AND CONCLUSIONS: The high incidence of arterial thrombotic events in our PV and ET patients was associated with a high incidence of stenosis detectable by arterial ultrasound. Hyperviscosity, endothelial damage due to leukocyte activation with subsequent thrombus formation, hyperhomocysteinemia and hyperexpression of activating genes such as JAK2 and STAT5 are all features characteristic of PV and ET that may contribute, along with other risk factors, to the development and progression of atherothrombosis. Cytotoxic treatment in PV and TE may be beneficial both through its antiproliferative effect on hematopoiesis and on the atherosclerotic plaques, atherogenesis being described as a proliferative disease of the vessel wall.

Pathogenic pathways in acute myeloid leukemias.

Despite the common clinical, hematological and prognostic features that define acute myeloid leukemia (AML) there is considerable heterogeneity among individual cases, suggesting different pathogenic pathways. Based on a simple theoretical model, according to the vital characteristics of the leukemic clone (proliferative rate and resistance to apoptosis) we propose a classification of AML into two broad categories: a) high leukemic clone vitality (HLV) AML, corresponding roughly to the World Health Organization (WHO) classification group of entities "AML with recurrent cytogenetic abnormalities" and b) low leukemic clone vitality (LLV) or "opportunistic" AML corresponding to the WHO groups "AML with multilineage dysplasia" and "therapy-related AML". HLV-AML leukemic clones are characterized by rate-limiting genomic mutations capable of conferring proliferation/survival advantage over a normal hematopoietic environment while in LLV-AML, the leukemic clones are not particularly proliferative or apoptosis-resistant, but are nevertheless selected against an impaired, previously damaged hematopoietic environment. Such a pathogenesis-oriented classification might have therapeutic and prognostic implications, providing a theoretical basis for a further adaptation of the current standard treatment strategies to the individual characteristics of the AML patients.

Hematopoietic stem cell transplantation in malignant lymphoproliferative diseases. A single center, retrospective study.

During the 1982-1997 period, out of the 133 hematopoietic stem cell (HSC) transplantations performed at Mont Godinne for malignant hemopathies, 85 were done in lymphoproliferative diseases: 27 in aggressive non-Hodgkin's lymphomas (ANHL), 21 in multiple myeloma (MM) patients, 17 in low-grade non-Hodgkin's lymphomas (LNHL), 9 in Hodgkin's disease (HD) cases, 9 in acute lymphoblastic leukemia (ALL) and 2 in chronic lymphocytic leukemia (CLL) patients. According to the HSC source, there were 19 allogeneic bone marrow grafts and 66 autologous HSC grafts (14 bone marrow and 52 peripheral stem cell grafts). In 40 cases the procedure was done after achieving partial or complete remission through conventional chemotherapy and in 45 patients in case of relapse or progressive disease. The conditioning regimens consisted of high-dose chemotherapy, associated with total body irradiation in case of allografts and in autografts for ALL. Engraftment was obtained in all autografted patients, transplant related mortality (TRM) occurring in 2 patients (3%). In the allogeneic transplantation group, 1 patient did not engraft and TRM was much higher, occurring in 10 cases (52%). Hematologic recovery occurred significantly faster in the autograft group than in the allograft group. The overall costs of an autograft were much lower than those of an allograft. Out of the 59 patients followed 4 years or longer, 23 (39%) are alive, free of disease, the proportion varying from 57% in the HD cases to 16% in the MM cases. The overall survival at 4 years was 49%. Negative prognostic factors for disease free survival at 4 years included male sex, lack of complete remission status at transplantation and MM diagnosis, whereas male sex and allografting were the negative prognostic factors for the 4-year overall survival.

Immunological methods in the diagnosis of monoclonal gammopathies. The prognostic impact of the immunological data.

Immunological data are extremely important for the diagnosis of monoclonal gammopathies (MG). During the 1977-1998 period, 160 MG cases were diagnosed in our laboratory. Out of the 130 multiple myeloma (MM) cases, 70 (53.85%) met the strictly immunological criteria; in the rest of the cases, the immunological data had to be corroborated with the bone marrow cytology and bone X-ray data. We tried to assess retrospectively the prognostic impact of the immunological data on the 60 cases that were treated and followed at the Hematology Dept. of the Cancer Institute, Cluj. The following parameters were found to have a favorable prognostic impact on survival: IgG monoclonal component, kappa light chains, response to therapy with plateau phase achievement, Salmon Durie stages I and II at diagnosis.

Granulocyte-macrophage colony stimulating factor treatment of febrile neutropenia following remission induction chemotherapy for acute leukemia.

Recombinant human granulocyte macrophage colony stimulating factor (GM-CSF) was given for 2-6 days in 10 patients with febrile neutropenias following remission induction chemotherapy for acute leukemia (6 acute lymphoblastic and 4 acute myeloid leukemias) in which, broad spectrum and targeted antibiotherapy was ineffective. In 7 patients, the addition of GM-CSF helped overcome the serious infectious complications, leading to an accelerated neutrophil recovery averaging 18.4 days, significantly faster than in a historic group of 9 similar patients in whom GM-CSF was not given Side-effects of GM-CSF treatment were minor and reversible. GM-CSF treatment did not have any significant effect on the achievement of complete remission status.

Plasma fibronectin in peripheral arterial disease.

When compared to values obtained in 24 age-matched normal weight normolipidemic control subjects, plasma fibronectin level was not significantly changed in the 28 patients with peripheral arterial disease, although endothelial cell-derived factor VIII related antigen was found to be obviously increased. On the other hand the 15 patients with obliterative atherosclerosis of the lower limbs who were hyperlipidemic (mainly hypertriglyceridemic) displayed a plasma fibronectin level (46.3 +/- 2.8 mg/dl) which was higher than the mean values recorded in both control subjects (36.2 +/- 1.66 mg/dl) and in normolipidemic patients with peripheral arterial disease (34.5 +/- 4.14 mg/dl). The results suggest that an increased level of plasma fibronectin should be connected rather to a peculiar pattern of hepatic protein synthesis occurring in overweight and hypertriglyceridemic subjects than to a release of this glycoprotein from an ongoing arterial lesion.

Serum lactate dehydrogenase and alkaline phosphatase activities and serum cholesterol level in bone marrow blood.

Serum cholesterol level as well as serum lactate dehydrogenase (LDH), alkaline phosphatase (ALP) and gamma-glutamyl transferase (GGT) were measured in 65 samples of bone marrow blood and in matched peripheral blood taken from patients with various hematological diseases. As expected, serum LDH activities were higher and serum total cholesterol levels were lower in the bone marrow blood than in the blood taken from the cubital vein. More interestingly, an important increase of heat-labile ALP, but not of serum GGT, was found in the bone marrow blood obtained from patients characterized by a proliferating bone marrow. Actually, both LDH and ALP activities were obviously higher in the bone marrow blood of patients with megaloblastic anemia, myelodysplastic syndrome and chronic myeloid leukemia than in samples taken from patients with chronic lymphocytic leukemia, a disease characterized by a slower proliferation rate. While the expected increased LDH activity is the result of an accelerated turnover of bone marrow cells implying the release of this enzyme from the dividing and/or decaying cells, the much higher activity of the heat-labile alkaline phosphatase found in the bone marrow blood would reflect an enhanced local remodeling of bone structures, probably related to an expanded proliferating bone marrow. The lower serum cholesterol level in the bone marrow blood could be subsequent to an enhanced uptake of low density lipoproteins by specific receptors on the bone marrow cells.

Plasma protein C and antithrombin III in patients with acute leukemia.

When compared to values recorded in the 32 healthy control subjects, plasma protein C activity was found to be significantly decreased in the 29 patients with acute leukemia and especially in those considered to be in a critical condition. On the other hand, plasma antithrombin III activity did not significantly differ from the values noted in control subjects. The concomitantly occurring high plasma fibrinogen levels and low serum cholinesterase activity were highly suggestive for a switch of hepatic protein synthesis towards the production of acute phase proteins. It is therefore considered that in the absence of a consumption coagulopathy, changes affecting plasma protein C and antithrombin III should be related to a modified pattern of hepatic protein synthesis.

Serum cholesterol and apoprotein B levels and serum cholinesterase activity in selected hematologic malignancies.

As compared to values recorded in 10 healthy normal-weight normolipidemic control subjects, serum cholesterol and apoprotein B levels as well as serum cholinesterase activity were found to be obviously decreased in the 28 patients with acute leukemia, the lowest levels being associated with the worst prognosis. The values of the above-mentioned biochemical variables in the 21 patients with chronic disorders (13 with chronic myeloproliferative disease and 8 with chronic lymphocytic leukemia) were not as low as in patients with acute leukemia. It should however be mentioned that in patients with chronic myelogenous leukemia, the lowest levels of serum cholesterol were correlated with a large tumor burden as assessed by a score taking into account for clinical and hematologic parameters. It is concluded that hypocholesterolemia could be regarded as a factor of adverse prognosis in hematologic malignancies, being probably the result of both enhanced catabolism of low density lipoproteins and impaired hepatic lipoprotein synthesis.

High-dose cytotoxic therapy with autologous bone marrow or peripheral blood progenitor cell transplantation in malignant lymphomas.

The present paper is an attempt to assess the efficiency of high-dose cytotoxic therapy followed by autologous bone marrow or peripheral progenitor cell rescue with hematopoietic growth factor support given in a group of 27 patients (16 men, 11 women) at the Department of Hematology of the Mont Godinne University Clinics, mainly in the same interval 1990-1994. The reasons for introducing such a therapy in these patients (6 with Hodgkin's disease, 14 with intermediate or high grade, aggressive non Hodgkin lymphomas and 7 with low grade follicular non Hodgkin lymphomas) were relapse of disease after conventional therapy (11 cases), resistance to initial therapy (5 patients) or because of histologically proven transformation to a more aggressive form (one case); in 10 patients with extended, poor prognosis forms, the procedure was used as part of the first line therapy. The conditioning high dose chemotherapy was given according to various regimens, most of them containing Cyclophosphamide, BCNU and Etoposide, with or without total body irradiation. In 14 patients, bone marrow (BM) graft was used, while peripheral blood progenitor cells (PBPC) were infused in the remaining 13 patients. The number of infused granulocyte-macrophage colony forming units (CFU-GM) ranged between 7,650 and 3,900,000/kg, with a mean value of 461,000/kg. The median time intervals required to reach an absolute neutrophil count > 500/microliter, a platelet count > 50,000/microliter and a hematocrit > 30% were 13 days, 20 days and 23 days respectively. Growth factors (GM-CSF and G-CSF) and PBPC use shortened the time for neutrophil recovery as well as neutropenia-related complications. No procedure-related death was observed and complete remission was achieved in 22 cases (81.4%); after a mean follow-up of 32.6 months, 14 patients (55.5%) are alive and free of disease, while in 7 patients (31% of the complete responders) relapse occurred at an average time interval of 8.2 months since the procedure.

Hepatitis B and C virus infection in Romanian non-Hodgkin's lymphoma patients.

We determined the hepatitis C virus (HCV) antibodies (anti-HCV) and the hepatitis B virus (HBV) surface antigen (HBsAg) in a cohort of 68 consecutive non-Hodgkin's lymphoma (NHL) patients diagnosed and treated in our institution between December 1997 and March 1999. 27 cases were diagnosed as low-grade, 33 as intermediate-grade, and eight as high-grade NHL. In 35 cases (51.4%) we found evidence of either HCV or HBV infection. Anti-HCV antibodies were found in 20 patients (29.5%) and HBsAg was found in 21 patients (30.8%). In six patients both anti-HCV and HBsAg were present. Anti-HCV were present in 12/27 low-grade NHL cases (44.4%) and in 8/41 intermediate/high-grade (aggressive) NHL cases (19.5%, P < 0.03). HBsAg was found in 10/27 low-grade NHL cases (37%) and in 11/41 aggressive NHL cases (26.8%). Evidence of liver disease, as reflected by elevated aminotransferases or typical alterations at liver biopsy, was present in eight patients. Cryoglobulins were present in six patients, all anti-HCV positive and with low-grade NHL. The prevalence of both HCV antibodies and HBsAg was significantly higher (P < 0.0001) in our NHL cases than in a sample of the general Romanian population, where the prevalence of anti-HCV was 4.9% and that of HBsAg was 6.3%. It is difficult to say whether either HCV or HBV had actually been involved in lymphomagenesis or if alpha-interferon treatment would be effective in this subset of patients.