Effects of clozapine on hematopoiesis and the cytokine system.

Using a bioassay for hematopoietic progenitor cells we looked for mechanisms causing clozapine induced neutropenia and agranulocytosis. Micro-agar-cultures of normal peripheral blood mononuclear cells (MNC) of eight patients currently treated with clozapine and of eight probands not receiving any kind of pharmacological treatment were incubated with increasing concentrations of clozapine (0, 7.5, 15, 30 micrograms/ml). Erythropoiesis and megakaryopoiesis were totally unaffected by clozapine. A biologically relevant suppression of granulopoiesis (CFU-GM) could only be shown in cultures incubated with 30 micrograms/ml clozapine. Cytokine analysis presented a strictly dose-dependent suppression of GM-CSF and neopterin release in all cultures. There was no difference between patients and controls at any clozapine concentration. The data support a possible role for cytokines as one mediator of the agranulocytosis producing effects of clozapine.

Recombinant interferon-alpha-2C in chronic myelogenous leukaemia: relationship of sensitivity of committed haematopoietic precursor cells in vitro (BFU-E, CFU-GM, CFU-Meg) and clinical response.

In an ongoing phase-II trial we aimed to predict clinical responsiveness of Philadelphia chromosome positive (Ph1+) chronic myelogenous leukaemia (CML) to recombinant IFN-alpha-2C (rIFN-alpha-2C) by pretesting in vitro. From five normal controls and 14 CML patients in chronic phase, bone marrow samples were taken before treatment and tested for antiproliferative activity by rIFN-alpha-2C, using a microagar culture system for BFU-E, CFU-GM, and CFU-Meg. Light-density nucleated bone marrow cells were stimulated for BFU-E and CFU-Meg colony formation with Alpha medium containing 20% serum obtained from a patient with severe aplastic anaemia. CFU-GM growth was induced with conditioned medium from the cell line GCT. In normal controls BFU-E, CFU-GM and CFU-Meg colony formation was inhibited by rIFN-alpha-2C in a dose-dependent manner. BFU-E proved to be the most sensitive cell lineage (IC50: 65; range: 53-116 U/ml) whereas CFU-GM was about 20 times less sensitive (IC50: 643; range: 480-897 U/ml). The sensitivity of CFU-Meg ranged between these two colony types with 50% growth inhibition at an IFN concentration of 160 (range: 68-246 U/ml). A heterogeneous response to rIFN-alpha-2C in vitro was seen in CML patients. Three of the 14 patients were 'resistant' to rIFN-alpha-2C in vitro with IC50 values for BFU-E, CFU-GM and/or CFU-Meg colony formation greater than 10(4) U/ml. Patients were subsequently treated with a daily dose of rIFN-alpha-2C of 5 x 10(6) U. Four patients achieved a complete and six achieved a partial haematological response. Of the four non-responders three rapidly progressed into blastic crisis. Thus it was seen that treatment failure to interferon was accompanied by IFN-resistance in vitro of BFU-E, CFU-GM and/or CFU-Meg colony formation by bone marrow precursors (p less than 0.01). These results suggest a predictive value of IFN-sensitivity testing in vitro in Ph1 + CML.

[Variability of coping strategies in coronary artery bypass surgery patients]. / Zur Variabilität des Coping bei Patienten mit aortokoronarer Bypass-Operation.

The state-versus-trait discussion in coping research would become more meaningful if attention is paid to the variability both of coping patterns in individual patients as well as stability of single coping strategies over time. 35 patients undergoing coronary artery bypass surgery were interviewed three times about their coping and stress experience: after cardiac catheterization, on the day before surgery, and six days after surgery. Anxiety and depression were measured. A coping attitude of "positive passivity" was present at all three points of time. While the group means were stable, vast interindividual differences occurred. Also, there was a high degree of scatter in the stability of single coping items; emotion related coping modes were more stable than cognition and action related ones. The variability of the patients' coping patterns correlated positively with the amount of stress experienced and with preoperative depression. Different coping strategies are linked to a different degree with personality traits, emotional coping modes revealing the closest connection. The variability of individual coping efforts might be linked to a personality disposition characterized by a vulnerability for stress and depressive reactions.

The acute-phase protein alpha 1-antitrypsin inhibits growth and proliferation of human early erythroid progenitor cells (burst-forming units-erythroid) and of human erythroleukemic cells (K562) in vitro by interfering with transferrin iron uptake.

We have previously shown that the hepatic acute-phase protein alpha 1-antitrypsin (alpha 1-AT) inhibits transferrin (tf) binding to its receptor (tfR) of human placental membranes. To evaluate the possibility that this interaction can explain the pathophysiology of the changes in iron metabolism in the course of chronic disease, subsequently leading to anemia in chronic disease (ACD), we examined the effect of alpha 1-AT on cells of the erythroid cell line. alpha 1-AT completely prevented tf binding to tfR on K562 human erythroleukemic cells and on reticulocytes. This inhibitory potency was dose-dependent and competitive, as proved in equilibrium saturation and kinetic studies. The cytokines interleukin-1 (IL-1), IL-6, and tumor necrosis factor alpha showed no such effect. Internalization of the tf-tfR complex was inhibited with alpha 1-AT in a dose-dependent manner. Furthermore, alpha 1-AT profoundly reduced the growth of K562 cells as well as their proliferation, albeit to a lesser degree. Growth of early erythroid progenitor cells (burst-forming units-erythroid) was significantly suppressed by alpha 1-AT, but no effect on the growth of late erythroid progenitor cells (colony-forming units-erythroid) was detected. These inhibitions of alpha 1-AT were seen in high physiologic concentrations attained in the course of acute-phase situations. These data show that alpha 1-AT might be a mediator of the changes in iron metabolism that are characteristic of clinical findings in the course of ACD.

Abnormal megakaryopoiesis in patients with myelodysplastic syndromes: analysis of cellular and humoral defects.

In 13 patients with myelodysplastic syndrome (MDS) mature and immature erythropoietic (CFU-E, BFU-E), granulopoietic (CFU-GM) and megakaryopoietic (CFU-Meg) colony formation from human bone marrow mononuclear cells was evaluated in a microagar culture system. All but three patients exhibited abnormal CFU-Meg. The defect of CFU-Meg paralleled the reduction of BFU-E, whereas CFU-GM number declined to a lesser extent. Not only the CFU-Meg number, but also the number of megakaryocytes (Mk) per colony was reduced suggesting an additional functional CFU-Meg defect. Megakaryocytic growth factor (Meg-CSF) abnormalities in MDS patients were detected using normal nonadherent T-lymphocyte depleted bone marrow cells as target cells for serum testing. Even for sera from patients with a reduction of platelets and bone marrow megakaryocytes Meg-CSF levels were not increased. No cellular or humoral inhibition could be detected in an MDS patient with a 5q- karyotype, who had an isolated defect of the megakaryocytic cell lineage at presentation. Some patients revealed a spontaneous formation of mixed erythrocytic, granulocytic and megakaryocytic clusters in the presence of fetal calf serum or autologous patient serum, probably representing autonomous proliferation of the malignant clone. In conclusion, both cellular and humoral factors can cause abnormalities of the megakaryocytic cell lineage in MDS patients.

Treatment of severe clozapine-induced neutropenia with granulocyte colony-stimulating factor (G-CSF). Remission despite continuous treatment with clozapine.

BACKGROUND: A 17-year-old boy suffering from a severe schizophrenic disorder of the paranoid type and mental retardation did not respond to treatment with typical antipsychotics, whereas under clozapine treatment he showed a favourable response. Discontinuation of clozapine led to an acute psychotic relapse. During clozapine treatment the patient developed severe neutropenia. METHOD AND RESULTS: Due to the history of unsatisfactory response to traditional antipsychotics, clozapine treatment was continued despite white blood cell (WBC) decline. Concomitant treatment with G-CSF was followed by a rapid normalisation of WBC. CONCLUSIONS: This case report is not intended to challenge the clinical practice of discontinuing clozapine upon the development of neutropenia/agranulocytosis, but rather to stimulate further research in the pathophysiology and clinical consequences of a clozapine rechallenge after a WBC decline, especially in patients with a rather complex symptomatology where no sufficient therapeutic results can be achieved with any other pharmacological intervention than clozapine.

A monoclonal antibody directed against the human intercellular adhesion molecule (ICAM-1) modulates the release of tumor necrosis factor-alpha, interferon-gamma and interleukin 1.

ICAM-1 is a cell surface glycoprotein which is one of the ligands for the leukocyte function-associated antigen (LFA-1). It is involved in leukocyte adhesion to endothelial cells as well as in immune functions requiring cell-cell contact. The quantitative expression of ICAM-1 in various cell types can be either induced or enhanced by treatment with cytokines, such as interferon-gamma (IFN-gamma), tumor necrosis factor (TNF)-alpha or interleukin 1 (IL 1), a phenomenon which results in the augmentation of binding to LFA-1-positive cells. In contrast, treatment with anti-ICAM-1 antibodies blocks this binding. A monoclonal antibody (mAb), termed 7F7, which recognizes an epitope on ICAM-1, was used to investigate the role of ICAM-1 in cytokine production by T lymphocytes and monocytes. Production of TNF-alpha. IFN-gamma and IL1 was significantly inhibited (p less than 0.01) by the incubation of mAb 7F7 with phytohemagglutinin-activated blood mononuclear cells (MNC) or isolated E rosette-positive T lymphocytes. The maximal level of inhibition was reached with 1 microgram/ml of purified antibody. A similar inhibition was obtained using saturating concentrations of 400 microliters/ml of mAb 7F7 hybridoma supernatant corresponding to an inhibitory activity of 1 microgram of purified mAb. In contrast, granulocyte/macrophage-colony-stimulating factor release showed a heterogeneous response over five experiments with an increase found in three experiments and a decrease in two experiments. Addition of increasing concentrations of supernatant or purified mAb to unstimulated MNC or T lymphocyte cultures had no effect on cytokine release. The observed inhibition of the production of TNF-alpha. IFN-gamma and IL 1 by antibody-mediated blockade of the ICAM-1 structure probably represents a negative circuit that serves to tune the activation of leukocytes and to avoid an overproduction of cytokines.

Recombinant human interleukin-8 restores function in neutrophils from patients with myelodysplastic syndromes without stimulating myeloid progenitor cells.

Prognosis in myelodysplastic syndrome (MDS) is not only correlated closely with blast cell count in bone marrow and chromosomal abnormalities but also correlated with decreased leucocyte count and function leading to acquisition of lethal infections. Recently, clinical trials in MDS have focused on the application of haemopoietic growth factors such as G-CSF or GM-CSF, which have proven to increase neutrophil count and function. However, these cytokines carry the risk of stimulating the malignant clone, particularly in patients with increased blast cell count. Therefore, investigation of cytokines which are able to stimulate neutrophil function without the potential risk of stimulating haemopoietic progenitor cells may be relevant for MDS. As the stimulatory effect of interleukin-8 on neutrophil function is well known, we investigated whether recombinant human IL-8 is also able to improve the function of neutrophils gained from patients with MDS. Using three different techniques--the E. coli killing assay (8 patients), the production of reactive oxygen as determined by cytochrome c reduction (7 patients) and chemiluminescence (8 patients)--a significant stimulation of neutrophil function at a concentration of 10 nm IL-8 was found in all test systems. No correlation with FAB classification was evident. On the other hand, IL-8 only mildly stimulated growth of myeloid progenitor cells in bone marrow culture of healthy individuals and MDS patients. This minimal stimulation was blocked by a neutralizing antibody directed against GM-CSF, suggesting an indirect effect of IL-8 via secondary GM-CSF release. Thus, IL-8 is able in vitro to repair the functional abnormalities of neutrophils from patients with MDS but has only a marginal influence on myeloid progenitor cells.

Correlation of minor histocompatibility antigen-specific cytotoxic T lymphocytes with graft-versus-host disease status and analyses of tissue distribution of their target antigens.

Peripheral blood mononuclear cells (PBMC) from 17 patients receiving HLA-identical sibling bone marrow grafts were stimulated with host pretransplant PBMC. Cytotoxic T-cell lines (TCL) with specificity for host pretransplant PBMC were obtained from 9 of these patients, all presenting with severe graft-versus-host disease (GVHD), but from none of the remaining cases lacking evidence of disease. Cytotoxic TCL were specific for host targets and failed to lyse donor cells. Monoclonal antibodies (MoAbs) blocking experiments and donor population screening analyses demonstrated that minor histocompatibility antigen (MiHA)-specific lysis of host targets was restricted by class I major histocompatibility complex (MHC) determinants. Whereas hematopoietic cells such as phytohemagglutinin (PHA) blasts or lymphoblastoid cell lines were susceptible to lysis by MiHA-specific TCL, keratinocytes (K) representing the natural targets of GVHD were quite resistant. Quantitative radioimmunometric measurements indicated very low constitutive expression of class I MHC antigens on K targets, which was readily increased by treatment with interferon-gamma (IFN-gamma). IFN-gamma treatment at the same time rendered these cells susceptible to lysis by MiHA-specific TCL. Host leukemic cells of 3 patients were recognized by MiHA-specific TCL in a chromium release assay and in one experiment host leukemic cells were effectively killed and their growth specifically inhibited in a leukemia colony assay by a clone. These data demonstrate that (1) host-specific cytotoxic TCL are detected exclusively in the PB of patients with acute GVHD grades II through IV after allogeneic matched bone marrow transplantation, and (2) their target antigens are simultaneously expressed on several host cell lines, including lymphoblastoid cell lines, PHA blasts, leukemic cells, and K. We also extend previous findings by showing that, besides the expression of the nominal MiHA, the density of the restricting class I MHC elements also crucially determines the extent of TCL lysis. Because of its capacity to enhance class I MHC antigen expression, IFN-gamma represents a key cytokine for determining the susceptibility of MiHA targets for lysis by TCL and clones, and in one patient an MiHA-specific clone recognized host leukemic cells and also inhibited host leukemic cell growth in a colony inhibition assay.