Matthias Zaiss

Whole blood analysis of reticulated platelets: improvements of detection and assay stability.

The flow cytometric analysis of reticulated platelets based on the fluorescent derivatization of their RNA content is increasingly used for the diagnostic classification of patients with thrombocytopenia as well as the monitoring of thrombopoiesis recovering under therapy. Many different modifications of the analytical protocol have been published following the first description in 1990 but consensus on the method has not yet been established. We have now reevaluated the assays methodology in order to optimize sensitivity and specificity and reduce the time length of incubation and washing procedures. In the modified experimental approach native whole blood is incubated for 15 min with an increased amount of thiazole orange (1 microg/ml) in the presence of phycoerythrin labeled antibodies directed against the constitutively surface expressed antigen GPlb. Data acquisition on the flow cytometer can be started immediately after stopping and stabilization of the reaction by paraformaldehyde fixation. Thiazole orange fluorescence was not significantly changed in thrombin-activated, degranulated platelets compared to resting platelets indicating no significant non-specific staining of platelet granules under the selected test conditions. In addition, experiments employing RNAse digestion demonstrated specificity of thiazole orange staining for platelet RNA.

Expression and Function of Homing‐Essential Molecules and Enhanced In Vivo Homing Ability of Human Peripheral Blood‐Derived Hematopoietic Progenitor Cells after Stimulation with Stem Cell Factor

Hematopoietic stem cell (HSC) homing from blood to bone marrow is a multistep process involving rolling, extravasation, migration, and finally adhesion in the correct microenvironment. With view to the hematopoietic recovery after clinical stem cell transplantation, we investigated the effect of stem cell factor (SCF) on the expression and the adhesive function of the α4β1 and α5β1 integrins very‐late antigen (VLA)‐4 and VLA‐5 on peripheral blood‐derived hematopoietic progenitor cells. After SCF stimulation, the expression of VLA‐4 and VLA‐5 on CD34+/c‐kit+ cells obtained from healthy donors increased from 54% to 90% and from 3% to 82%, respectively. For patient‐derived cells, the increase was 67% to 90% and 12% to 46%. The proportion of mononuclear cells adhering to the fibronectin fragment CH296 increased by stimulation with SCF from 14% to 23%. Accordingly, functional studies showed an approximate 30% increase of adherent long‐term culture‐initiating cell. The improvement of the homing abilities of SCF‐stimulated HSC was confirmed by transplantation into sublethally irradiated nonobese diabetic‐scid/scid mice. Six weeks after the transplantation, in eight of eight animals receiving human HSC with the addition of SCF, a profound multilineage hematopoietic engraftment was detected, whereas in the control group receiving only HSC, none of eight animals engrafted. Our data provide the first in vivo evidence that stimulation with cytokines improves the homing ability of transplanted human hematopoietic progenitor cells.

Autologous tandem transplantation: almost complete reduction of neutropenic fever following the second transplantation by ex vivo expanded autologous myeloid postprogenitor cells

Summary:Reduced post-transplant performance status because of infectious complications is still a problem following autologous peripheral blood stem cell transplantation (aPBSCT). In this study, a tandem transplantation scheme for 15 patients with breast cancer including etoposide (1500 mg/m2), ifosfamide (12 g/m2) and carboplatin (1500 mg/m2) as conditioning regimens, followed by aPBSCT, was used to evaluate the potential clinical benefit of the additional retransfusion of low numbers of ex vivo expanded committed myeloid postprogenitor cells (PPCs) (median 408 × 103 CFU-c/kg BW, range 0.93–1995) following the second transplantation. Following a 7+2 days expansion (using recombinant human SCF, IL-1β, IL-3, IL-6 + G-CSF), CFU-c generated from CD34-positive cells from leukapheresis products could be expanded by a median factor of 153 (range 5–434). Flow cytometric analysis and morphology of CFUs have shown a nearly exclusive expansion and differentiation of committed myeloid progenitor cells and a significant reduction of CD34-positive cells. In an intra- and interindividual comparison it could be shown that the retransfusion of committed myeloid postprogenitor cells significantly accelerates myeloid recovery. Although retransfusion of PPCs fails to abrogate severe neutropenia following aPBSCT, it significantly ameliorates infectious complications and shortens the duration of hospital stay.

CD84 expression on human hematopoietic progenitor cells

OBJECTIVE CD84 is a member of the CD2 subgroup of the immunoglobulin receptor superfamily. Members of this family have been implicated in the activation of T cells and NK cells. Expression of CD84 was originally described on most mononuclear blood cells as well as platelets. To elucidate its presence on other blood cell types, we analyzed the expression pattern of CD84 on human immature CD34+ and mature hematopoietic cells. METHODS Expression analysis was carried out by flow cytometry. The differentiation potential of CD84+ progenitor cells was assessed by colony-forming assays and long-term cultures. RT-PCR was used to analyze CD84 mRNA isoforms. RESULTS In addition to monocytes, macrophages, B cells, and some T cells, CD84 is expressed on the cell surface of the majority of granulocytes. In addition, 64%+/-5% of CD34+ progenitor cells isolated from peripheral blood and 30.5%+/-5% from bone marrow of healthy volunteers also express CD84. The majority of CD34+ cells coexpressing lineage antigens were CD84+. In methylcellulose CD34+CD84+ cells formed primarily erythroid colonies, whereas myeloid or mixed colonies were scarce. The frequency of long-term culture-initiating cells in peripheral blood was approximately fivefold higher in CD34+CD84- vs CD34+CD84+ cells. In short-term cultures, 95% of the initially CD34+CD84- cells became CD84+ after 72 hours. CONCLUSIONS CD84 is expressed on cells from almost all hematopoietic lineages and on CD34+ hematopoietic progenitor cells. The proliferative potential of CD34+ cells decreases with increasing CD84 expression, suggesting that CD84 serves as a marker for committed hematopoietic progenitor cells.

Differentiation of hematopoietic progenitor cells towards the myeloid and B-lymphoid lineage by hepatocyte growth factor (HGF) and thrombopoietin (TPO) together with early acting cytokines.

Abstract:  Objectives: The effect of stem cell factor (SCF), flt3‐ligand (FL), and interleukin (IL)‐3 (SF3) in combination with hepatocyte growth factor (HGF), thrombopoietin (TPO), and Hyper‐IL‐6 on maintenance and differentiation of early human peripheral blood‐derived progenitor cells was investigated. Methods: Single sorted CD34+ 38− cells were cultured with various combinations of these growth factors in order to identify the most effective cytokine combination. Then, lineage‐depleted cells were stimulated for 7 d in bulk culture before they were assessed by flow cytometry and in functional assays. Results: The highest number of clones in the single‐cell assay was obtained after culture with SF3 + TPO + HGF. Cell expansion with SF3 + TPO + HGF yielded an increase of the total cell number (11‐fold), the number of CD34+ cells (sevenfold), colony forming cells (CFC; 13‐fold), granulocytes (CD15/66b+; 45‐fold) and B‐cells (CD19/20+; 55‐fold). However, the number of long‐term culture initiating cells (LTC‐IC) decreased from 779 ± 338 per 1 × 105 CD34+ cells on day 0 to 253 ± 115 on day 7. In parallel, the number of pluripotent mouse repopulating cells decreased by the factor 11, and no significant change in the proportion of human myeloid or lymphoid cells found in the mouse bone marrow was noted. Conclusion: The observation that mature cells of different lineages are generated and that transplantable multipotent hematopoietic cells are lost during culture suggests the differentiation of early hematopoietic progenitors toward lineage committed cells by the tested cytokines. The detection of cells expressing B‐lymphoid markers after culture indicates a possible role in the propagation of B‐cells.

Activation of lymphocytes inhibits human monocyte to macrophage differentiation

Tissue macrophages (MAC) differentiate from circulating blood monocytes (MO) during a maturation step that is of crucial importance for their functional competence. In vitro a similar process of maturation can be observed, if MO are cultured in the presence of serum. In the work presented here, we show that activated lymphocytes can interfere with MAC differentiation. Resting lymphocytes have only marginal influence upon MO to MAC transition in vitro. However, if cells are activated by the lectins PWM or ConA or by double-stranded RNA (polyinosinic-polycytidylic acid, pI:C), normal MAC maturation is suppressed: MO stay small and do not acquire MAC maturation-associated surface molecules like carboxypeptidase M (CPM, determined by antibody MAX.1) or CD84 (determined by antibody MAX.3). This phenomenon can be induced by small numbers of lymphocytes and can be transmitted by soluble factors in cultures stimulated with ConA or PWM. IFN-gamma is present in these conditioned media and partially suppresses MAC maturation but cannot fully substitute for the conditioned media. On the contrary, in pI:C stimulated cultures, suppression of MAC differentiation is dependent on cell-cell contact. In conclusion, activated lymphocytes are able to suppress the terminal differentiation of MAC by several pathways depending on the mode of lymphocyte stimulation.

A multimodal treatment approach including high-dose chemotherapy in very advanced gastric cancer: evidence for control of metastatic disease

Summary:The present multimodal treatment approach was designed to achieve prolonged tumor control in advanced gastric cancer. A total of 26 patients with stage IV gastric cancer (metastatic disease n=25), ECOG performance status 0–3 and laparoscopically evaluated peritoneal status received a modified EAP schedule to prove chemosensitivity and to mobilize autologous peripheral blood stem cells (aPBSC). Patients without progressive disease proceeded to tandem high-dose chemotherapy (HD-CT) and aPBSCT. Patients with >50% reduction of the target lesion received a second cycle of HD-CT. Responders were selected for local R0 resections (D2 resection) according clinical criteria. Of 26 patients, 20(77%) achieved partial remission after dose-intensive chemotherapy: local R0 resection was achieved in 12 out of 14 patients selected for surgery (46% of all patients). Eight of these R0-resected patients initially had peritoneal carcinomatosis. With a median follow-up of 3.2 years, four patients are still alive. The median overall survival was 8.4 months (CI 2.5–14.4 months), for histologic regression grade 3 (seven out of 25 patients, 28%) 29 months (CI 12–46 months). The combined treatment approach is tolerable and feasible in advanced disease and opens a therapeutic window for a significant proportion of patients, even in cases with histologically proven peritoneal carcinomatosis.

Transplantation Strategies for High Risk Acute Myeloid Leukemias

Outcome of high risk leukemias as defined by unfavorable cytogenetics at the time of diagnosis is poor in almost all large trials on induction therapy of AML. Complete remission rates are in the range of 50–60%, and longterm survival after chemotherapy alone is reported to be lower than 10–15%. These data strongly suggest that transplantation strategies in 1st CR or PR are needed in this particular subgroup of patients. As recent studies adressed longterm outcome following autologous and allogeneic stem cell transplantation in pts with unfavorable cytogenetics, we will summarize these conflicting and still disappointing results and discuss potential problems as well as actual approaches to overcome major obstacles.

Peripheral blood stem cell transplantation during postremission treatment of de novo acute myelogenous leukemia

Intensification of induction and consolidation treatment in de novo AML lead to a continuous improvement of disease-free survival in the last 20 years. To increase dose-intensity during consolidation, patients in continued CR (n = 16) which did not qualify for allogeneic BMT, received busulfan/cyclophosphamide conditioning and peripheral blood stem cell transplantation (PBSCT) after double induction treatment with TAD (thioguanine, daunorubicin and AraC) and HAM (high-dose AraC and mitoxantrone) and an early consolidation with a second course of TAD. G-CSF treatment was started on day two after each chemotherapy as well as on the day of PBSCT. PBSC were collected after mobilization with HAM for back-up and after the second course of TAD for transplantation. PBSCT could be performed in all patients being in continued CR (1.33 × 106 CD34+ cells per kg (range 1.0 to 2.9 × 106); 1.38 × 105 CFU-GM/kg (range 0.14 to 2.8 × 105)) except one patient with persistent pancytopenia after the second course of TAD. A further mobilization with cyclophosphamide was necessary in three patients after the second course of TAD. Leukocyte counts of 109/1 were achieved within 10.4 days (range 8–12). In five cases the increase in thrombocyte counts was biphasic but remained stable > 100 × 109/1 after 59 days (range 16–92) in all patients. Severe toxicities (grade IV) were observed in 4 patients (peumonia 2 ×, pericarditis 1 ×, VOD 1 ×). One patient died of pneumonia. Relapse-free survival was 66% (95% confidence interval 58 to 74%) at a mean observation time of 27 months. Cytogenetically patients were found to be at good (n = 1) intermediate (n = 14) and high risk (n = 1). Of the four patients relapsing three only achieved CR after a second induction cycle, one was at high risk cytogenetically and two had an excessively high initial LDH level (> 1000 U/l). One patient developed a myelodysplastic syndrome. Thus, myeloablative chemotherapy with PBSCT has tolerable toxicity and may be performed routinely as consolidation treatment following TAD-HAM-TAD. The risk of relapse seems to be increased if achievement of CR is delayed. The observed relapse-free survival justifies a randomized trial with standard versus high-dose chemotherapy combined with the transplantation of in vivo purged PBSCs.