We thank the patients and their families for their trust in taking part in this study. The study was academically funded and supported by the Medical University Vienna, the General Hospital Vienna, and the Research Center for Molecular Medicine (CeMM) of the Austrian Academy of Sciences. We gratefully acknowledge funding from the Vienna Science and Technology Fund (LS16-034 to GSF and UJ), the Austrian Science Fund (F4704-B20 to PV, F4711-B20 to GSF, and P27132-B20 to PBS), and the European Molecular Biology Organization Long Term Fellowship (1543-2012 to GIV, 733-2016 to TP). BS acknowledges

No abstract

The hematopoiesis-specific G protein alpha subunit G alpha16 was shown to be expressed in early normal and malignant hematopoietic cell lines and has been suggested to play an important role in signal transduction of hematopoiesis. We previously demonstrated a strict correlation of G alpha16 mRNA and CD34 antigen expression in peripheral blood stem cells (PBSC). In PBSC mobilization, both markers are detectable at the time of hematopoietic recovery and progenitor cell release. In this study the possible use of G alpha16 determination in peripheral blood samples for monitoring patients undergoing stem cell transplantation was investigated. Normal peripheral blood is negative for G alpha16 expression. In all five patients G alpha16 mRNA expression appeared shortly before the time of blood cell recovery. When tested together with CD34 (three cases) a pattern different from CD34 antigen expression was found, reflecting a different mechanism of action. In two cases with different time points of leukocyte and platelet recovery G alpha16 mRNA was detected at both time points but not in the interval, thus suggesting a role of G alpha16 in multipotent precursor cells. CD34 mRNA tested in three patients was not detected at any time; this argues for different regulation of CD34 and G alpha16 mRNA. G alpha16 may be used as an indicator of hematopoietic recovery after autologous stem cell transplantation, suggesting that there are cell type-specific G protein-mediated signal transduction pathways of early hematopoiesis.

G proteins play an important role in signal transduction from cytokine receptors to intracellular effectors via different pathways, eg involving tyrosine kinases. In our previous studies, we demonstrated that mRNA expression of the hematopoiesisspecific G protein ␣-subunit G␣16 is a sensitive marker indicating the appearance of early myeloid and lymphoid progenitors. This study was designed to investigate cytokine effects on hematopoiesis in vivo and in vitro as reflected by G␣16 expression and sensitivity to the hemoregulatory peptide (pEEDCK)2 which harbors a structural homology to the effector domain of G␣16. Investigations on blood samples from lymphoma patients undergoing salvage therapy with different cytokine support showed that monitoring of the expression of G␣16 mRNA which appears to play a role in cytokine signalling via tyrosine kinases was a valuable complementation to CD34 screening for analyzing hematopoietic recovery after chemotherapy. We demonstrated that in contrast to CD34 which is only expressed in quiescent cells, G␣16 transcription occurs independently of cell cycle state. In vitro, we could show that G␣16 was also a valuable marker for confirming the immature state of ex vivo expanded blood stem cells from patients. A further part of the study was focused on the response of G␣16 and CD34 expressing cells to the granulocyte-derived hemoregulatory peptide (pyroGlu-Glu-Asp-Cys-Lys)2 = (pEEDCK)2 which harbors a G␣16-homologous sequence motif. Results obtained from in vitro assays which involved estimation of colony outgrowth from CD34-positive cells showed that the effect of (pEEDCK)2 on CD34 cells enhanced the effect of IL-3 or SCF. These data indicate that G␣16 may co-operate with (pEEDCK)2 in triggering the cytokine response of immature hematopoietic cells.

Human long-term bone marrow cultures (HLTBMCs) are a valuable in vitro model for studying the role of the haemopoietic microenvironment. Here we report the spontaneous appearance of EBV-positive B cells in 6/40 HLTBMCs from patients with various haematological diseases after 3-5 months of culture.After subcultivation of these cells, a novel type of cell line could be characterized, which displayed surface markers and morphological features typical for EBV transformed B-cell lines.As the deproteinized and ultrafiltrated culture supernatants of these cell lines were found to contain an agent with stroma toxic properties, they were termed SSB linesThe supportive function of bone marrow stroma for growth regulation of haemopoietic progenitors implicates a role of human long-term bone marrow culture (HLTBMC) as a model for investigating the close link between the stromal microenvironment and haemopoiesis (Gordon, 1994).Studies of HLTBMCs indicate that stroma cells mediate their effects on differentiation and proliferation of haemopoietic progenitors through cell-cell interactions (Gordon, 1988) and the secretion of soluble mediators (Metcalf, 1989).However, stromal cell lines have been described which provide a microenvironment facilitating proliferation of neoplastic and virus-infected cells (e.g. Mannick et al, 1987). Evolution of B-cell lines which are latently infected with the human B-cell lymphotropic herpesvirus (stroma-toxic-agent-secreting B-cell lines). This agent also exhibited a colony-inhibitory activity on in vitro myelopoiesis and erythropoiesis. These properties are typical for the two polyamines spermine and spermidine which were detected at elevated levels in the culture supernatants of SSB lines.The hypothesis that latent presence of EBV in bone marrow may induce an increased synthesis of spermine and spermidine, which are known to be associated with malignant haematological diseases and bone marrow aplasia, is discussed.