Peter Bettelheim

Cell Surface Structures on Human Basophils and Mast Cells: Biochemical and Functional Characterization

Publisher Summary This chapter gives an overview of cell surface membrane structures expressed on human mast cells and human basophils, with special respect to similarities of and differences between these cells. Mast cells and basophils are specialized effector cells of the immune system. Both cells express high-affinity immunoglobulin E (IgE) binding sites and play an important role in host defense mechanisms and allergic reactions. Mast cells and basophils synthesize and store large amounts of pro-inflammatory mediator molecules, and on activation, these mediators may be released to the extracellular space. The chapter provides the characterization of cell surface membrane structures expressed on human mast cells and human basophils. By the use of monoclonal antibodies to leukocyte differentiation antigens, the cell surface membrane antigen profile has been defined for both types of cells. Molecular and functional analyses of cell membrane antigens expressed on human blood basophils and human mast cells are performed. Thus, a number of cell surface receptors for basophil/mast cell growth factors and activating polypeptides, complement binding sites, receptors for immunoglobulins, recognition molecules, cell surface enzymes, and glycolipids are identified. Many of these surface membrane antigens play a very important role in inflammatory and allergic disease states that involve blood basophils and tissue mast cells. Mast cells and basophils share many phenotypical and functional properties. Despite this, striking differences exist. The cell surface phenotype documents that both cells are different and respond to different signals.

Standardization of flow cytometry in myelodysplastic syndromes: a report from an international consortium and the European LeukemiaNet Working Group

Flow cytometry (FC) is increasingly recognized as an important tool in the diagnosis and prognosis of myelodysplastic syndromes (MDS). However, validation of current assays and agreement upon the techniques are prerequisites for its widespread acceptance and application in clinical practice. Therefore, a working group was initiated (Amsterdam, 2008) to discuss and propose standards for FC in MDS. In 2009 and 2010, representatives from 23, mainly European, institutes participated in the second and third European LeukemiaNet (ELN) MDS workshops. In the present report, minimal requirements to analyze dysplasia are refined. The proposed core markers should enable a categorization of FC results in cytopenic patients as ‘normal’, ‘suggestive of’, or ‘diagnostic of’ MDS. An FC report should include a description of validated FC abnormalities such as aberrant marker expression on myeloid progenitors and, furthermore, dysgranulopoiesis and/or dysmonocytopoiesis, if at least two abnormalities are evidenced. The working group is dedicated to initiate further studies to establish robust diagnostic and prognostic FC panels in MDS. An ultimate goal is to refine and improve diagnosis and prognostic scoring systems. Finally, the working group stresses that FC should be part of an integrated diagnosis rather than a separate technique.

Further Characterization of Surface Membrane Structures Expressed on Human Basophils and Mast Cells

Recently, we were able to establish the immunologic surface marker profile of human basophils and mast cells. In the present study, the characterization of these cell types was extended by the use of monoclonal antibodies (mAbs) to hemopoietic differentiation antigens. Basophils and mast cells were enriched by mAbs and complement from chronic myeloid leukemia blood (n = 5) and dispersed lung tissue (n = 4), respectively. A panel of 80 mAbs was tested for being reactive with purified cell populations using flow cytometry and/or a combined toluidine blue-immunofluorescence staining procedure. In addition to previous findings, basophils were found to react with mAbs directed against the 126-kilodalton dipeptidylpeptidase IV (CD26), platelet glycoprotein IIa (CD31), CD40 antigen known to share sequence homology with nerve growth factor receptor, leukosialin (CD43), CD44 antigen, the ICAM-1 antigen (CD54) and VIM2-reactive gangliosides involving the sialofucooligosaccharide sequence (CDw65L). Bsp-1 was found to be a specific marker for human basophils, whereas mast cells were not stained by this reagent. Basophils apparently lack CD22 antigen, gangliosides detected by CDw65 mAbs (except CDw65L) and CD71 antigen (transferrin receptor). Mast cells were found to express CD43 and CD44 antigen. In contrast, mast cells lack CD22, CD26, CD31, CD40 and CDw65 antigen. These results provide further evidence that both blood basophils and mast cells express a unique immunologic surface marker profile including binding sites for a variety of immunomodulating ligands and adhesion molecules.

VIL-A1, a monoclonal antibody reactive with common acute lymphatic leukemia cells.

The VIL-A1 monoclonal antibody raised against Reh cells reacts with common acute lymphatic leukemia (CALL) cells but not with normal or malignant B or T lymphocytes. It also shows no binding to normal or malignant myeloid, monocytic or erythroid cells, nor does it react with thrombocytes. The antibody is of IgM class and lyses CALL cells very efficiently in the presence of rabbit but not human complement. Immunoprecipitation experiments followed by SDS-polyacrylamide gel electrophoresis under reducing conditions revealed that VIL-A1 defines a 95,000 mol. wt membrane protein. Approximately 40% of it binds to lens culinaris lectin. Capping experiments showed that the membrane component defined by VIL-A1 co-caps with the one recognized by another recently described monoclonal antibody to CALL cells (J5).

Cell lineage heterogeneity in blast crisis of chronic myeloid leukaemia

Summary Blast cells from 45 patients with chronic myeloid leukaemia in blast crisis (CML‐BC) were immunologically phenotyped with a panel of 26 monoclonal antibodies and studied for terminal deoxynucleotidyl transferase (TdT) content. Out of 45 blast‐populations, 28 showed a myeloid, 14 a lymphoid, two a mixed and one an unclassifiable marker profile.

Prognostic impact of karyotype and immunologic phenotype in 125 adult patients with de novo AML

One hundred-twenty-five adult patients with de novo acute myeloid leukemia (AML) were treated according to a standard 7 + 3 induction regimen. Karyotype and immunological phenotype of blasts examined prior to treatment were correlated with each other, with response to treatment and duration of survival. The following monoclonal antibodies (mAbs) were used for immunological phenotyping: VIM-D5 (CD15), MY7 (CD13), MY9 (CD33), VIM-2 (CDw65), VIM-13 (CD14), 63D3 (CD14), VID-1 (anti HLA-DR), WT1 (CD7), CLB-Ery3 (antiblood group H antigen), C17-27 (CD61), and an antiserum against TdT. Despite a considerable overlap between the individual groups, patients with specific aberrations as defined by the MIC classification (n = 39) showed distinct, characteristic, myeloid or myelomonocytic immunophenotypes. In M2/t(8;21) there was a significant association with negativity to CD13, in M3/t(15;17) with negativity to CD15 and HLA-DR, whereas in M4/inv(16) expression of blood group H antigen was unexpectedly found. The response to therapy, as well as rate of complete remission as duration of survival, was better in patients with M2/t(8;21), M3/t(15;17), and M4Eo/inv(16) as compared to all other patients and significantly worse in patients with M5a/t/del(11)(q23). In 35 patients with normal karyotype and 16 patients with cytogenetic anomalies not presently associated with FAB subtypes the expected correlations of rather immature myeloid immunologic phenotypes with M1 and M2 morphology and CD14 expression in monoblastic leukemias was found. Remission rate and survival were significantly worse in 19 patients with complex nonrandom aberrations, where blast cell expression of blood group H antigen and of TdT were significantly increased.

First-line treatment of Waldenström's disease with cladribine

Abstract Purpose: To assess the activity and side effects of cladribine (2-CdA) treatment in patients with advanced Waldenströms disease. Patients and methods: Ten symptomatic patients without prior therapy were included in a prospective multicenter trial. 2-CdA was administered daily at 0.12 mg/kg body weight in a 2-h i.v. infusion over 5 consecutive days; this was repeated every 28 days for four cycles. Patients achieving a remission received interferon alfa-2c (IF) 15 μg s.c. three times a week for 1 year. Results: All 10 patients responded to 2-CdA (100%; 95% confidence interval, 68–100%), with one complete (CR) and eight partial responders (PR); one patient had only one 2-CdA cycle and showed a minor improvement (MR). Patients tolerated the treatment well. Despite considerable immunosuppression, an infection occurred in only two patients. After a median observation period of 57 weeks, three patients had shown progression, including one who died of lymphoma. Conclusion: 2-CdA induction and IF maintenance is a well-tolerated therapy for symptomatic untreated patients with advanced Waldenströms disease and offers excellent palliation.

Revisiting guidelines for integration of flow cytometry results in the WHO classification of myelodysplastic syndromes—proposal from the International/European LeukemiaNet Working Group for Flow Cytometry in MDS

Definite progress has been made in the exploration of myelodysplastic syndromes (MDS) by flow cytometry (FCM) since the publication of the World Health Organization 2008 classification of myeloid neoplasms. An international working party initiated within the European LeukemiaNet and extended to include members from Australia, Canada, Japan, Taiwan and the United States has, through several workshops, developed and subsequently published consensus recommendations. The latter deal with preanalytical precautions, and propose small and large panels, which allow evaluating immunophenotypic anomalies and calculating myelodysplasia scores. The current paper provides guidelines that strongly recommend the integration of FCM data with other diagnostic tools in the diagnostic work-up of MDS.

Granulocyte colony-stimulating factor (G-CSF) as an adjunct to induction chemotherapy of adult acute lymphoblastic leukemia (ALL)

SummaryOur purpose was to evaluate the ability of re-combinant human granulocyte colony-stimulating factor (r-metHuG-CSF) as an adjunct to induction chemo-therapy of acute lymphoblastic leukemia (ALL) to ameliorate chemotherapy-induced neutropenia and thus allow patients to receive full doses of chemotherapy on time. Sixteen consecutive patients with adult ALL (13 de novo, three relapsed) were treated with induction chemo-therapy according to the BMFT protocol and received in addition r-metHuG-CSF (200μg/m2/day). Patients who were treated with the same induction chemotherapy but without G-CSF between 1982 and 1990 served as controls. Fifteen of the 16 patients achieved complete hematological remission. One patient died because of fungal septicemia. Compared with historical controls, G-CSF-treated patients had a significantly faster neutrophil recovery in phase I, resulting in neutrophil counts > 1000/μl at day 17 vs day 26 (in median) in controls. In phase II, the onset of severe leukocytopenia (< 1500/μl) was significantly (p = 0.01) delayed and the degree of leukocytopenia less pronounced (mean nadir 3300/μl) in G-CSF-treated patients compared with controls (1880/μl). The number of days of febrile neutropenia was not different in phase I. In phase II it was lower in study patients (0 vs 1.1 days), but the difference did not reach statistical significance (p = 0.09). Full doses of chemo-therapy could be given on time to 11/13 (85%) G-CSF pa-tients but to only 7/30 (23%) controls. These data indicate that (a) G-CSF can be given along with chemotherapy in induction treatment of ALL without compromising efficacy; (b) the duration of neutropenia in phase I is markedly shortened and the degree of leukocytopenia in phase II ameliorated; (c) these beneficial effects allow patients to receive full doses of chemotherapy on time.

The growth and differentiation of mast cells.

Mast cells (MCs) are local immune cells involved in host defense mechanisms and allergic response. They usually develop from MC committed progenitor cells which in turn are derived from uncommitted hemopoietic stem cells. MC precursors are supposed to develop in the bone marrow (bm) cavities as well as in extramedullary tissues. MC precursor cells also have the potential to circulate in the blood stream. After homing in the tissues they give rise to mature MCs. Recruitment and differentiation as well as terminal maturation of MCs is regulated by a complex network of factors. Two major arms of control have been delineated based on in vitro studies and experimental animal models. The first involves the response of the progenitor cells to growth inducing cytokines, such as IL-3. This type of control promotes the generation of MC precursor cells. The second arm of control involves the microenvironmental network interacting with the MC progenitors. It consists of both stroma cell- and immune cell-derived differentiation factors and the direct interaction of cells. It may be important for homing of MC progenitors during embryogenesis and probably throughout life. The stromal component also determines terminal differentiation towards a particular type of MCs and also supports in vitro development of MCs in long term cultures. Growth and function of the mature MCs in the various tissues may be triggered by additional factors including the interactions of MCs with other leukocytes and nerve cells. The coupling of MC activation processes with subsequent proliferation may be a triggering factor in allergic disease. This article attempts to provide a synthesis of current knowledge on MC development.(ABSTRACT TRUNCATED AT 250 WORDS)