Autologous del(20q)-positive erythroid progenitor cells, re-emerging after DLI treatment of an MDS patient relapsing after allo-SCT, can provide a normal peripheral red blood cell count.

A 54-year-old RhD-negative male with del(20q)-positive myelodysplastic syndrome was transplanted with bone marrow from an HLA-identical RhD-positive sibling donor. Cytogenetic relapse was detected 21 months after stem cell transplantation (SCT), with reappearance of the original del(20q)-positive clone and reversion to recipient RhD-negative blood group. The patient received sequential donor lymphocyte infusions (DLIs), resulting in mild graft-versus-host disease and pure red cell aplasia. At 2 years post DLI, the patient remains in a stable condition, despite a dominance of recipient-derived erythro- and granulopoiesis originating in del(20q)-carrying progenitor cells. We conclude that reappearance of autologous erythropoiesis, upon relapse after allogeneic SCT, may be predictive of erythropenia after DLI and that re-emerging autologous del(20q)-positive erythropoiesis post DLI can provide a normal peripheral red blood cell count. Furthermore, in patients relapsing after blood-group-mismatched transplantation, a possible reversion to recipient blood group should be considered prior to blood transfusion or DLI.

Treatment of anemia in myelodysplastic syndromes with granulocyte colony-stimulating factor plus erythropoietin: results from a randomized phase II study and long-term follow-up of 71 patients.

Treatment with erythropoietin (epo) may improve the anemia of myelodysplastic syndromes (MDS) in approximately 20% of patients. Previous studies have suggested that treatment with the combination of granulocyte colony-stimulating factor (G-CSF) and epo may increase this response rate. In the present phase II study, patients with MDS and anemia were randomized to treatment with G-CSF + epo according to one of two alternatives; arm A starting with G-CSF for 4 weeks followed by the combination for 12 weeks, and arm B starting with epo for 8 weeks followed by the combination for 10 weeks. Fifty evaluable patients (10 refractory anemia [RA], 13 refractory anemia with ring sideroblasts [RARS], and 27 refractory anemia with excess blasts [RAEB]) were included in the study, three were evaluable only for epo as monotherapy and 47 for the combined treatment. The overall response rate to G-CSF + epo was 38%, which is identical to that in our previous study. The response rates for patients with RA, RARS, and RAEB were 20%, 46%, and 37%, respectively. Response rates were identical in the two treatment groups indicating that an initial treatment with G-CSF was not neccessary for a response to the combination. Nine patients in arm B showed a response to the combined treatment, but only three of these responded to epo alone. This suggests a synergistic effect in vivo by G-CSF + epo. A long-term follow-up was made on 71 evaluable patients from both the present and the preceding Scandinavian study on G-CSF + epo. Median survival was 26 months, and the overall risk of leukemic transformation during a median follow-up of 43 months was 28%. Twenty patients entered long-term maintenance treatment and showed a median duration of response of 24 months. The international prognostic scoring system (IPSS) was effective to predict survival, leukemic transformation, and to a lesser extent, duration of response, but had no impact on primary response rates.

Aggressive course of primary plasma cell leukemia with unusual morphological and cytogenetic features.

A case of aggressive plasma cell leukemia with unusual morphological and cytogenetic features is reported. A 65-year-old man was admitted to hospital due to anemia, thrombocytopenia, and renal insufficiency. Bone marrow examination and peripheral blood smear revealed a large number of pleomorphic cells with convoluted and multilobulated nuclei. Immunohistochemistry of the bone marrow biopsy was negative for anti-keratin antibodies CAM.5.2 and AE1/AE3, but positive for EMA. The immunophenotypic features of these cells were suggestive of plasma cell origin with positivity for CD38, CD56, CD9, and CD44 and a weak positivity for CD71 and CD45 (40% of the cells), while all other markers of hematopoietic origin were negative. Furthermore, a serum protein electrophoresis showed a monoclonal component type IgG-kappa of 70 g/l. The cytogenetic analysis demonstrated a hypotetraploid clone with multiple numerical and structural abnormalities. Although some of the aberrations found are associated with plasma cell malignancies--e.g., structural rearrangement of chromosome 1, del(6q), and monosomy 13--the karyotypic complexity in the present case is unusual. The course of the disease was very aggressive, and the patient died 3 days after admission.

A combination of granulocyte colony-stimulating factor and erythropoietin may synergistically improve the anaemia in patients with myelodysplastic syndromes.

In an attempt to obtain a synergistic effect on the hemoglobin levels in anaemic patients with myelodysplastic syndromes (MDS), granulocyte colony-stimulating factor (G-CSF) and erythropoietin (epo) were combined in a clinical phase II trial. Twenty-two patients with MDS were included in the study. G-CSF was given alone for six weeks and then in combination with epo for the following twelve weeks. Eight (38%) of 21 evaluable patients showed a significant increase in hemoglobin. One patient with a previous response and subsequent failure to epo alone improved after the addition of G-CSF. Responses were more frequent in patients with less advanced pancytopenia, lower endogenous levels of serum-epo and in those with ring sideroblasts in the bone marrow. The response frequency of 38% is higher than in any study of epo as monotherapy. Moreover, patients with ring sideroblasts, who respond poorly to epo alone, showed a response rate of 60%. Our findings suggest a synergistic in vivo effect of granulocyte-CSF and erythropoietin in patients with myelodysplastic syndromes.

A pilot study of piperacillin and ciprofloxacin as initial therapy for fever in severely neutropenic leukemia patients.

We studied the efficacy of piperacillin and ciprofloxacin as initial parenteral therapy in 41 adult patients with leukemia who developed 47 febrile episodes during severe neutropenia following chemotherapy. 40 patients (98%) survived their febrile episode(s), whereas 1 patient died of infection. When assessed at 72 h after initiation of treatment (early evaluation), 24/47 episodes (51%) had been successfully treated. These 24 favourable responses were seen in 15/24 (63%) microbiologically documented infections and 9/19 (47%) fever of unknown origin (FUO). At the resolution of fever (late evaluation) 46 episodes were evaluable, and 28 (61%) had responded successfully to piperacillin and ciprofloxacin. Successful treatment was most frequently observed in microbiologically defined infections, 18/23 (78%). Three of 5 (60%) Gram-positive, 11/12 (92%) Gram-negative and 1 of 2 mixed bacteremias were successfully treated. In contrast, only 10/19 (53%) FUO and none of 4 clinically defined infections had responded. Thus, this pilot study indicates that piperacillin and ciprofloxacin may be a safe and effective combination for the treatment of febrile episodes in severely neutropenic leukemia patients, which merits further investigation in randomized trials.

Bernard-Soulier syndrome in two Swedish families: effect of DDAVP on bleeding time.

We present 2 patients with Bernard-Soulier syndrome from two different families. The parents of one of the patients were found to have had common ancestors in the 17th century. The platelet membrane content of glycoprotein (GP)Ib was measured in the patients and their first-degree relatives with an ELISA technique based on monoclonal antibodies. Both patients had very low levels of GPIb. In one of the families the heterozygotes had reduced expression of GPIb but in the other the obligate heterozygotes had normal values, suggesting that the molecular pathology differs between the two families. In both patients, bleeding time was shortened by infusion of DDAVP (1-deamino-8-D-arginine vasopressin), although it was not completely normalised. DDAVP may be of some therapeutic value in cases of Bernard-Soulier syndrome.

Treatment of myelodysplastic syndromes with retinoic acid and 1 alpha-hydroxy-vitamin D3 in combination with low-dose ara-C is not superior to ara-C alone. Results from a randomized study. The Scandinavian Myelodysplasia Group (SMG).

63 evaluable patients with myelodysplastic syndromes (MDS) and 15 with acute myelogenous leukemia (AML) were randomized between low-dose ara-C (arm A) and low dose ara-C in combination with 13-cis-retinoic acid (13-CRA) and 1 alpha-hydroxy-vitamin D3 (1 alpha D3) (arm B). 69 patients were evaluable and 18 (26.1%) responded to therapy. The addition of 13-CRA and 1 alpha D3 had no positive influence on survival of the patients, remission rates or duration of remissions. 12/27 patients in arm A and 6/29 patients in arm B progressed from MDS to AML during the course of the study (p = 0.0527). Arm B gave significantly more side-effects than arm A (p = 0.005). Therapeutic effects of 13-CRA and 1 alpha D3 on MDS is not supported by this study. However, an inhibiting effect on AML development in some MDS subgroups cannot be excluded.

Fulminant course of infectious mononucleosis with virus-associated hemophagocytic syndrome.

A fatal case of infectious mononucleosis due to serologically verified Epstein-Barr virus infection in a previously healthy 30-year-old man is presented. The clinical course was characterized by severe prostration, persistently high spiking fever, and continuous development of enlarged lymph nodes. Hematologic examination revealed peripheral leukopenia and thrombocytopenia, and in the bone marrow an increased number of benign histiocytes showed marked hemophagocytosis. At autopsy abnormal lymphoid infiltrates were present in several tissues. The pathogenesis of this infection-associated hemophagocytic syndrome is discussed in terms of the possibility of an impaired immune response to infectious agents.

Biochemical properties of the eosinophil cationic protein and demonstration of its biosynthesis in vitro in marrow cells from patients with an eosinophilia.

The eosinophil cationic protein (ECP), which has been shown to be secreted both in vitro and in vivo, is a cytotoxic unique constituent of eosinophil granules. To increase the understanding of the mechanisms behind the role of the eosinophil as a cytotoxic effector in disease, a detailed biochemical characterization of ECP was performed. A considerable molecular heterogeneity was revealed when purified ECP was eluted isocratically from a high-resolution cation exchange resin; the separation, reproducibly achieved, of five components was probably due to hydrophobic interaction with the resin. These polypeptides, which reacted quantitatively with anti-ECP antiserum, showed molecular weights (mol wt) of 19,500 and 16,700 and showed almost identical amino acid compositions. The amino-terminal sequence for one of the polypeptides was (in the standard one-letter code) (R-P-X-Q-F-T-R-A-Q-W-F-A-I-Q-H-I-S-L-N-P-R-R-C-T-I-A-M-R-A-I-N-N-Y-). The biosynthesis of ECP was demonstrated in marrow cells from patients with eosinophilia using labeling with (14C)-leucine, followed by immunoprecipitation with anti-ECP, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and fluorography for visualization of labeled ECP. Biosynthesis was demonstrated of mol wt 22,000 ECP, which may represent precursor ECP, since with time some of it was processed into ECP with a mol wt of 18,000 to 19,000. Monensin, a proton ionophore, blocked the processing of mol wt 22,000 ECP. This study shows that ECP consists of a family of similar polypeptides. These may, however, have different biological activities.

The biosynthesis of neutrophil and eosinophil granule proteins.

Composition of azurophil and specific granules from human polymorphonuclear neutrophils and granules from eosinophils is presented. Biosynthesis of the granule proteins is discussed in detail with particular emphasis on neutrophil myeloperoxidase (MPO) and eosinophil cationic protein (ECP).

Effect of zinc and other cations on the release of the eosinophil cationic protein.

The eosinophil cationic protein, ECP, is a unique eosinophil granule constituent, which is released extracellularly after exposure of the eosinophils to a non-phagocytosable surface such as complement-coated Sephadex beads. The ECP is released to some extent even in the absence of Ca2+ and Mg2+, though both these cations augment the release reaction tested alone, and an optimal release is observed only in the presence of 2 mmol/l Ca2+ and 2 mmol/l Mg2+ in the medium. Zn2+ at concentrations from 0.25-4.0 mmol/l inhibited the release of ECP in a dose-dependent fashion, with or without Ca2+ and Mg2+ in the medium. Mn2+ had dual effects, stimulating the ECP release in the absence of Mg2+ and Ca2+, and inhibiting the release in the presence of these cations. Li1+ caused minor inhibition of ECP release, but only in the absence of Ca2+ and Mg2+. The inhibitory effect of Zn2+ was immediate and reversible after washing of the cells, suggesting that the inhibition is due to interaction with the plasma membrane functions.

Purification of eosinophil peroxidase and studies of biosynthesis and processing in human marrow cells.

Human eosinophil peroxidase (EPO) was purified from leukocytes obtained from a patient with hypereosinophilia. EPO was extracted from the granule fraction using 0.2 mol/L sodium acetate pH 4.0, and the extract was subjected to gel chromatography on Sephadex G-75 and ion exchange chromatography on Biorex 70. The mol wt calculated from gel chromatography was approximately 50,000. However, under reducing and denaturing conditions, polyacrylamide gel electrophoresis revealed two subunits with mol wt of 50,000 and 15,000. The biosynthesis of EPO was studied in marrow cells from patients with eosinophilia using labeling with (14C)-leucine, followed by immunoprecipitation with anti-EPO, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and fluorography for visualization of labeled EPO. Biosynthesis of an Mr 53,000 subunit was demonstrated. Biosynthetic labeling of the Mr 15,000 subunit was not demonstrated. A labeled Mr 25,000 chain was detected and may represent a degradation product or a chain that, after further modification, produces the Mr 15,000 subunit. Labeling was also detected in two polypeptides with mol wt of 78,000 and 72,000. These forms of EPO seem to represent precursor polypeptides subjected to proteolytic processing in a similar manner as has been reported for myeloperoxidase (MPO). However, Monensin, a proton ionophore, which blocks the processing of MPO, did not inhibit processing of EPO, indicating separate mechanisms by which MPO and EPO are directed to granules.

Mechanisms for adherence of eosinophils to an antibody-coated surface.

Eosinophils may act by degranulation after attachment to a surface. As the mechanisms of adherence are not understood, we have investigated the dependency on Fc(IgG) receptors and other mechanisms by studying the adherence of human eosinophils to albumin-Sepharose beads coated with either specific rabbit IgG antibody, F(ab')2 antibody fragments or serum under different conditions. Adherence to Sephadex beads and albumin-coated microtitration plates was also investigated. 50% of the eosinophils adhered spontaneously to all 3 different surfaces not coated with the antibody, whereas only 25% of neutrophils and less than 10% of mononuclear cells adhered. A small but significant increase in adherence to albumin-Sepharose or albumin-coated plastic occurred after addition of the IgG-antibody, but not after addition of F(ab')2 fragments, indicating that the Fc region was responsible for some increase in adherence. Incubation of eosinophils with IgG-Fc fragments prevented the additional antibody-mediated adherence. As Fc receptor-negative eosinophils adhered almost as well as Fc receptor-positive cells, it appears that the Fc receptors are of minor importance and instead, a nonspecific adherence mechanism, possibly unique for the eosinophil, seems to be the most important in eosinophil adherence to antibody-coated surfaces.

Mechanisms for eosinophil degranulation; release of the eosinophil cationic protein.

Mechanisms for degranulation in human eosinophils were evaluated. Release of eosinophil cationic protein (ECP), a unique eosinophil granule constituent, was measured upon exposure of purified eosinophils to a large surface consisting of Sephadex beads coated with serum, which leads to complement activation. Extracellular release of approximately 15% of the cellular ECP occurred both with eosinophils from patients with eosinophilia and normal people. Almost all eosinophils isolated from patients with eosinophilia and normal people adhered to serum-treated Sephadex. The data suggest that interaction through C3 receptors is a prerequisite for ECP release from eosinophils when exposed to serum-treated Sephadex. Both cytochalasin B, cytochalasin D and hydrocortisone reduced the release of ECP. Neither the cytochalasins nor hydrocortisone inhibited the adherence of eosinophils to the Sephadex beads. Thus the inhibitory effect of these agents on ECP release is a direct effect on the degranulation process. ECF-A, histamine and colchicine did not affect the release mechanism. No direct relationship was found between degranulation and oxidative burst inasmuch as some soluble mediators induced a high respiratory burst without a concomitant ECP release. Our data suggest that mechanisms for degranulation are not fully identical in eosinophils and neutrophils.

Altered density, metabolism and surface receptors of eosinophils in eosinophilia.

A comparison was made between eosinophils from normal persons and patients with eosinophilia. Highly purified eosinophils were obtained by centrifugation in a Percoll density gradient. Studies were carried out on density distribution, oxygen consumption upon adherence to serum-coated Sephadex and expression of cell surface receptors for IgG and complement. In eosinophil leukaemia the density of eosinophils was abnormally low. Abnormal light density fractions of blood eosinophils were also detected in the hypereosinophilic syndrome (HES). Light density eosinophils of HES showed morphological signs of degranulation consonant with the finding of a low content of granular eosinophil cationic protein (ECP) suggesting degranulation in the circulation or abnormal granule formation in the marrow. In addition, such cells exhibited a higher oxygen consumption than eosinophils with normal density upon adherence to serum-coated Sephadex. Low density eosinophils showed a greater number of cells with Fc-IgG and complement receptors than high density cells. Likewise exudate eosinophils displayed an abnormally low density with higher than normal oxygen consumption indicating that eosinophils may be activated in the tissues. In one patient with HES, a febrile episode resulted in a disappearance of eosinophils with a normal density while abnormal low density eosinophils increased. Our findings suggest that eosinophils from some patients with eosinophilia may be 'activated' in the circulation.

Variations of cationic proteins from eosinophil leukocytes in food intolerance and allergic rhinitis.

Challenge tests were performed in patients with food intolerance and allergic rhinitis to evaluate the usefulness of measurement of the eosinophil cationic protein (ECP) of serum to distinguish different allergic reactions. In one group of patients with food intolerance symptom-induced challenge resulted in a marked decrease of serum-ECP. The number of blood eosinophils decreased simultaneously in some but not all of these patients. In another group of patients with food intolerance serum-ECP displayed peak-like increases followed by a decrease. The decrease in serum-ECP may reflect that consumption of ECP is a result of idiosyncrasy in the target organ. In allergic rhinitis some patients showed an initial peak-like increase of serum-ECP, which was abolished by pretreatment with disodium-cromoglycate but not by pretreatment with antihistamine. Similar results have previously been demonstrated for allergic asthma. The difference obtained in serum-ECP upon challenge in typical reagin-mediated allergy and food intolerance may indicate that the latter is not reagin-mediated. However, the interpretation of data is difficult because of lack of knowledge regarding the turnover in the circulation of ECP.