The effect of antiviral therapy on t(14;18) translocation and immunoglobulin gene rearrangement in patients with chronic hepatitis C virus infection.

The mechanism of lymphomagenesis of hepatitis C virus (HCV)-related B-cell lymphoma is unknown. Recently, it has been suggested that HCV may induce B-cell clonal proliferation and t(14;18) translocation in patients chronically infected with the virus. Thus, this study investigated the effect of antiviral treatment on immunoglobulin heavy-chain gene (IgH) rearrangement and t(14;18) translocation in HCV infected patients. Twenty-nine patients with chronic HCV infection were studied in whom IgH rearrangement and/or t(14;18) translocation were previously detected. The IgH rearrangement (FR3/JH) and t(14;18) translocation (MBR bcl2-JH) were detected in peripheral blood mononuclear cells by polymerase chain reaction. Fifteen of 29 patients (8 with IgH rearrangement, 6 with t(14;18) translocation, and 1 with both) were treated with either interferon-alpha or by combination therapy with interferon and ribavirin for 6 to 12 months. IgH rearrangement became negative in 7 of 9 treated patients compared with only 1 of 8 of nontreated patients (P <.02). The t(14;18) translocation became negative in 6 of 7 treated patients compared with 1 of 6 nontreated patients (P =.03). Disappearance of IgH rearrangement or t(14;18) translocation was strongly associated with virologic response to treatment. Two t(14;18)+ patients developed B-cell lymphoma during follow-up. Antiviral treatment appears to be effective in eliminating the clonal proliferation of B cells in patients with chronic HCV infection and may prevent the subsequent development of lymphoma. The mechanism can be related to a direct effect of interferon-alpha on the proliferating clone or to an indirect effect by eradicating the antigenic stimulus.

bcl-2 and immunoglobulin gene rearrangement in patients with hepatitis C virus infection.

An association between chronic hepatitis C virus (HCV) infection and clonal proliferation of B cells, including B cell lymphoma, has recently been demonstrated. However, the mechanism of malignant transformation is still unknown. It has been shown that B cells from patients with type II mixed cryoglobulinaemia (MC), strongly express the antiapoptotic bcl-2 oncogene product. Therefore, we investigated a possible mechanism of lymphomagenesis, the occurrence of bcl-2 and immunoglobulin gene rearrangement (IgH) in HCV-infected patients. Three groups of patients were studied: (1) 44 patients with HCV and MC (anti-HCV and HCV RNA positive); (2) 59 patients with chronic HCV infection without MC; (3) 50 patients with chronic liver disease (CLD) not related to HCV infection. The t(14;18) translocation (MBR bcl-2-JH) and IgH rearrangement (FR3/JH) were detected by polymerase chain reaction (PCR) in peripheral mononuclear cells. bcl-2 translocation was detected in 17/44 (39%), 7/59 (12%) and in none of the patients of groups 1, 2 and 3 respectively (P < 0.01). Monoclonal IgH rearrangement was detected in 15/44 (34%), 5/59 (8.5%) and 2/50 (4%) patients of groups 1, 2 and 3 respectively (P < 0.05). HCV-infected patients had a higher prevalence of monoclonal IgH rearrangement and bcl-2 translocation than patients with CLD of other aetiologies. These data suggest that HCV may play a role in the multistep mechanism of lymphomagenesis by inducing clonal proliferation of B cells and inhibition of apoptosis.

Immune and hematopoietic reconstitution after transplantation of cord blood progenitor cells: case report and review of the literature.

Transplantation using umbilical cord progenitor cells as the source of the stem cells is increasingly recognized as another form of allogeneic transplantation with curative intent. However, the different patterns of hematopoietic and immunological reconstruction have been described in very few patients. A 20-month-old boy presented with acute leukemia. He received standard AML induction and consolidation therapy, after which he underwent allogeneic transplantation using HLA-matched sibling stem cells obtained from the umbilical cord. The preparative regimen consisted of busulfan and cyclophosphamide. White cell recovery, despite concomitant use of G-CSF, was slow, reminiscent of the engraftment pattern without the use of growth factor. Erythroid recovery was best recorded using fetal cell HbF level. Platelet transfusion independence occurred on day +31. Immunologic reconstitution revealed an early NK cell recovery by 6 weeks and progressive T cell recovery until 3 months, with continued increase in counts thereafter. However, the CD4/CD8 ratio remained low even at 14 months post-transplantation. Recovery of B cells was slower until day +120. Proliferative response was within normal range on day +120. This report describes the unique engraftment pattern following umbilical cord blood transplant and emphasizes the pattern of immunological and hematological reconstitution.

Cryohemolysis for the detection of hereditary spherocytosis: correlation studies with osmotic fragility and autohemolysis.

Laboratory methods aimed to assess the presence of spheroidal cells such as osmotic fragility, autohemolysis, and glycerol lysis time are very elaborate, time consuming, and often give inconclusive results. We have developed a diagnostic test based on a unique sensitivity of HS cells to hypertonic cryohemolysis and analyzed blood samples of 55 HS patients. The patients were divided into two subgroups, clinically affected probands and their relatives. To get quantitative comparisons with the classic methods, the cryohemolysis results were compared to two parameters of the osmotic fragility test: the salt concentration that causes 50% hemolysis, and the percent lysis at a constant salt concentration. Autohemolysis results were also compared. To evaluate which of these tests has the best analytical power, we calculated the mean results and 2 SDs of each parameter in a control group, and then looked to see which of them was best in identifying the patients. The cryohemolysis test was the single parameter that identified all cases including asymptomatic carriers of the disease. The ability of this test to identify the less severe cases probably reflects the dependency of the cryohemolysis on factors that are more related to the primary membrane molecular defects and less by the surface area to volume ratio.

Compound heterozygosity for two alpha-globin gene defects, Hb Taybe (alpha 1; 38 or 39 minus Thr) and a poly A mutation (alpha 2; AATAAA-->AATAAG), results in a severe hemolytic anemia.

We have identified two alpha-globin gene variations in an Arabian male with severe hemolytic disease through sequencing of amplified DNA of his alpha 2- and alpha 1-globin genes. One of the abnormalities involves a CAC (ACC or CCA) deletion between codons 36 and 41 of the alpha 1-globin gene. This leads to the synthesis of an abnormal alpha chain with one instead of two threonine residues at positions 38-39 and to the formation of the unstable Hb Taybe. The second variation is a mutation located in the poly A site of the alpha 2-globin gene (AATAAA-->AATAAG) which is common among Arabian people. Family studies have shown that the two variations are located on opposite chromosomes. The hemolytic disease in this man, resembling Hb H disease, is likely the result of a severe downregulation of both alpha-globin genes on the chromosome with the alpha 2 poly A mutation, and the instability of the alpha-Taybe chain being the product of an alpha 1-globin gene; this leaves only one alpha 2-globin gene normally active.

The role of electrostatic forces in the interaction between the membrane and cytoskeleton of human erythrocytes.

Evidence is presented that electrostatic forces play a major role in the interaction between the cell membrane and cytoskeleton of human erythrocytes. Experiments were carried out on the effects of ionic strength variation, Ca2+ and Mg2+ ions, dimethonium ion and lipophilic ions on the release of spectrin from the erythrocyte ghost. In addition it was shown that the release of spectrin for fixed Ca2+ or Mg2+ concentration shows a maximum as a function of Na+ concentration. All results are consistent with the existence of a repulsive electrostatic force between membrane and cytoskeleton.

A simple spectroscopic method for studying erythrocyte ghost resealing.

A novel spectroscopic method is described for following the kinetics of resealing of hemolysed erythrocyte ghosts. The procedure is based on the broadening of the EPR spectrum of nitroxyl radicals by paramagnetic ions. The method is used to study the effect of Ca2+, Mg2+ and dimethonium ion on the kinetics of resealing.

EPR study of the hydrophobic interaction of spectrin with fatty acids.

The hydrophobic interaction between spin-labelled stearic acid and spectrin was studied by electron paramagnetic resonance (EPR) and fluorescence quenching. The results are quantitatively interpreted in terms of two types of binding site on spectrin. A comparison between the results of the EPR and fluorescence experiments show the drawback of the fluorescence method in binding studies.

Hypertonic cryohemolysis: a diagnostic test for hereditary spherocytosis.

Red blood cells of subjects with hereditary spherocytosis are specifically susceptible to temperature changes while suspended in hypertonic solutions. Based on this property, a new diagnostic test for hereditary spherocytosis is presented. The suggested method is 100% sensitive in the diagnosis of all patients, including asymptomatic obligate carriers with hereditary spherocytosis, and is very specific in different control groups. Unlike other methods designed for the diagnosis of hereditary spherocytosis, this test does not depend on the cells' surface- area-to-volume ratio. Normal red blood cells that were induced to become spherocytes by different means (i.e., vinblastine, lysophosphatidyl choline, and heat) and showed increased osmotic fragility did not become susceptible to the hypertonic cryohemolysis conditions.

Spectrin modifications in a heterozygous case of both hereditary elliptocytosis and beta-thalassemia.

The clinical and hematological parameters of a patient described here, who inherited the genes of both hereditary elliptocytosis (HE) and beta-thalassemia, seem to reflect a mutual enhancement of the two diseases. The coexistence of the two pathologies is probably also responsible for the observed changes in spectrin: the appearance of an extra spectrin band between tetramers and dimers on denaturing gel electrophoresis and the metabolic-dependent reduction in spectrin amount. It is assumed that the instability of the skeletal network that results from the HE pathology caused increased exposure of the spectrin molecule to oxidative damage that usually occurs in thalassemic red cells. The products of such oxidation may have led to abnormal spectrin associations which finally resulted in the above changes.

Direct involvement of spectrin thiols in maintaining erythrocyte membrane thermal stability and spectrin dimer self-association.

Human erythrocytes vesiculate upon exposure to temperatures of 49 degrees C and above. Pretreatment of the cells with the thiol-alkylating agent N-ethylmaleimide (NEM) lowers the temperature needed to produce the same effect. Concomitant with the cells' heat susceptibility, skeletal mechanical instability and an increase in spectrin dissociation have been reported (Smith and Palek (1983) Blood 62, 1190). In the present study, similar results were achieved by preincubation of the cells with diamide, which could be reversed by reduction with dithiothreitol. Another oxidative agent, sodium tetrathionate, could only induce the temperature susceptibility, with little effect on spectrin dissociation. Incubation of spectrin solutions with NEM or diamide caused decreased association of spectrin dimers and increased dissociation of spectrin tetramers. Estimation of membrane and spectrin thiols in the treated cells showed that NEM was effective while blocking less than 20% of the thiols. Diamide and tetrathionate blocked more than 50% of the thiols, but were less effective than NEM. It is suggested that some very defined population of thiols is essential for spectrin self-association and for membrane thermal stability. They are more available to NEM than to diamide and less so to tetrathionate. Other thiols participate in maintaining the membrane thermal stability only.

Sialic acid of platelets and granulocytes in polycythemia vera.

Previous studies have shown that erythrocytes of polycythemia vera (PV) patients have an increased content of sialic acid on their membrane compared with normal erythrocytes. This has been attributed to the presence of an abnormal clone known to proliferate in this disease. Since granulocytes and platelets originate from the same stem cell that is involved in this disease, it was of interest to see whether these cells also bear increased amounts of sialic acid compared with their normal counterparts. While normal values were found for granulocyte populations, elevated values were found for the platelet populations of PV patients.

Some characteristics of circulating erythrocytes in polycythaemia vera: common features with normal young and foetal red blood cells.

Although it is well established that in polycythaemia vera (PV) both 'normal' and 'abnormal' erythroid progenitors proliferate, it is less known to what extent the circulating erythrocytes express normal characteristics. We found reduced erythrocyte densities, decreased MCHC, and increased lipid content. These properties, together with increased sialic acid, seem to explain the extremely low sedimentation rate and decreased deformability of polycythaemic blood samples. Other characteristics were high activity of glycolytic enzymes, increased in vitro production of lactate, and a concomitant decline in ATP and 2,3-diphosphoglycerate. Some of these properties have been described in foetal erythrocytes and are features of normal young red cells. However, they seem to represent true features of PV and not a consequence of younger cell populations. The similarity between mature erythrocytes in PV and in foetal life supports the possibility that the proliferation process in this disease has a mechanism in common with foetal erythropoiesis.

Hypertonic cryohemolysis of pathologic red blood cells.

Human erythrocytes suspended in hypertonic solutions undergo hemolysis when the temperature of the suspension is changed from 37 degrees C toward 0-4 degrees C. It has been suggested that the hypertonic environment causes some proteins of the skeletal network to be changed in such a way that their normal adaptation to temperature changes is prevented, thus resulting in cryohemolysis. In the present study, we compared the cryohemolysis of some pathologic red blood cells in hypertonic sucrose and NaCl to normal cells. Erythrocytes of hereditary spherocytosis (HS) were found to be significantly more fragile than all others in hypertonic sucrose, while they behaved normally in hypertonic NaCl. In contrast, erythrocytes of thalassemic patients showed decreased susceptibility to cryohemolysis, both in hypertonic sucrose and in NaCl. Autoimmune hemolytic anemia samples behaved like normal samples, both in NaCl and in sucrose. The erythrocytes of congenital dyserythropoietic anemia-type II patients showed two types of cryohemolysis; one pattern was similar to that of HS, and the other one presented normal levels in sucrose and reduced levels in NaCl. The different patterns of cryohemolysis described for the pathologic cells are thought to reflect different lesions in the membranes of the erythrocytes of the various hemolytic disorders. It is hoped that studying the cryohemolysis of abnormal red cells may contribute some illumination as to molecular interactions in intact cells in health and in disease.

Glucose-6-phosphate isomerase deficiency-Nahariya: extreme in vitro and in vivo lability of the mutant enzyme.

A glucose-6-phosphate isomerase deficiency is described in an Arab boy suffering from chronic hemolytic anemia. The patient was probably true homozygous for the defect. The residual enzyme activity in his red blood cells (RBC) was approximately 30% of normal. The most striking enzyme abnormality observed was an extreme heat lability: upon incubation at 45 C, greater than 90% of activity was lost within 15 min. Furthermore, an increased affinity for the substrate glucose-6-phosphate was shown. The lability of the enzyme was also shown to exist in vivo by separating the patient's RBC into four fractions of different cell age by centrifugation on a discontinuous density gradient. This in vivo lability of the enzyme is believed to be the main cause of the hemolytic diathesis. Remarkably, the residual activity of the enzyme in the RBC of obligate heterozygotes was comparable to that in the patient. However, their enzyme activity was only slightly more labile than that in normal RBC and consequently no signs of hemolysis were noticed.

Glucose-induced hemolysis of spheric red blood cells in hereditary spherocytosis: new aspects of the autohemolysis test.

Increased autohemolysis with a partial protecting effect of added glucose is a common finding in hereditary spherocytosis (HS). For unknown reasons, in some rare cases, glucose fails to prevent the increased autohemolysis (Type II autohemolysis). The authors investigated the autohemolysis of such a patient and found that glucose actually induced the hemolysis prior to energy depletion. Old erythrocytes proved to be more fragile in the presence of D-glucose than did young ones. Other D-hexoses reacted similarly to D-glucose, while L-glucose did not. After being splenectomized, the patient's red blood cells' specific sensitivity to D-hexoses disappeared unless a 24-hour preincubation of his whole blood was performed. Other HS blood samples also became fragile in the presence of D-glucose if a 24-hour preincubation at 37 degrees C was made. Normal blood samples preheated to 50 degrees C or normal washed red blood cells in hypotonic conditions also showed a glucose-induced hemolysis. The authors assume that during the routine autohemolysis test, the accumulation of the hexose is followed by volume expansion and results in hemolysis of those cells with extremely low surface area-to-volume ratio.

Moving boundary electrophoresis of red cells on differently treated polycythaemic patients.

Red cells of polycythaemia vera (PV) patients have a significantly higher rate of electrophoresis than red cells of normal controls and stress polycythaemia patients. The highest increment in the electrophoretic velocity was noted for PV patients treated with phlebotomy or hydroxy urea. The red cells of PV patients treated with 32P and those that progressed to myeloid metaplasia showed a normal rate of electrophoresis. We assume that the increased negative charge found on the red cells of PV patients is typical of the abnormal clone proliferating in this disease. The normal electrophoretic velocity found for the red cells of the 32P treated patients or those that progressed to myeloid metaplasia might indicate that the circulating red cells of these patients are no longer descendants of the abnormal stem cell.

Unique profile for erythrocyte membrane acetylcholinesterase in hereditary spherocytosis.

Acetylcholinesterase of human erythrocytes from healthy donors and from patients with hematological disorders was analysed in a search for differential membrane parameters. Two substrates were used to estimate the exposure of acetylcholinesterase active site in the membrane: phenylacetate, a hydrophobic substrate, to determine total enzyme activity, and acetylcholine, an ionic substrate, to measure the externally reactive enzyme. The sensitivity of acetylcholinesterase to added stearic acid was also analysed. Three categories of the disorders studied were discerned: (a) The erythrocyte acetylcholinesterase profile was indistinguishable from normal control in beta-thalassemia minor and groups of patients with autoimmune hemolytic anemia or congenital dyserythropoietic anemia type II. (b) A marked decline in acetylcholinesterase with both substrates and reduced sensitivity to stearic acid were exhibited by the erythrocytes of paroxysmal nocturnal hemoglobinuria, beta-thalassemia major and other autoimmune hemolytic anemia and congenital dyserythropoietic anemia type II patients. Normal erythrocytes, either aged or pretreated to 50 degrees C, also showed similar characteristics. (c) Hereditary spherocytosis was singly differentiated by an elevated acetylcholinesterase activity with acetylthiocholine and by a vastly diminished sensitivity to stearic acid, while activity with phenylacetate was equal to control. This distinct profile may reflect the unique organization of the erythrocyte membrane in hereditary spherocytosis.

Moving boundary electrophoresis and sialic acid content of normal and polycythaemic red blood cells.

Moving boundary electrophoresis has been used for the assessment of the negative charge on erythrocyte membranes. The equipment is easy to construct and simpler to use than the microelectrophoresis technique. There is a direct relationship between the electrophoretic velocity measured by the moving boundary method and the sialic acid content of the red cell membrane. Polycythaemic erythrocytes have a higher electrophoretic mobility and higher sialic acid content than normals. Separation of the erythrocytes according to their densities shows that old polycythaemic erythrocytes have a higher electrophoretic mobility and sialic acid content than young normal ones. This suggests that the difference is not associated with a younger erythrocyte population in polycythaemia vera, but may represent a membrane change in the abnormal erythroid clone present in this disease.