Gram-positive bacteria causing life-threatening septicaemia in neutropenic patients undergoing bone marrow transplantation represent a major transplantation-related complication. We therefore evaluated teicoplanin for suspected gram-positive infections after an inadequate response to initial empiric beta-lactams and aminoglycoside combination therapy. Eleven patients included in this regimen received an allogenic (five patients) or autologous (six patients) bone marrow transplant for acute myeloid leukemia (AML), Non-Hodgkin-Lymphoma (NHL, high grade) or other malignant diseases. All patients under study developing a primary septicaemia of unknown origin (15 patients) or a catheter-related septicaemia (one patient) were treated with teicoplanin, 400 mg i.v. once daily in combination with a cephalosporin and an aminoglycoside (ceftazidime 2 g i.v., t.i.d., and 400 mg netilmicin i.v., q.d.). Teicoplanin serum levels were monitored closely of all patients under investigation.All 16 patients responded to therapy, 15 patients were clinically cured, one patient improved under therapy. So far we have not observed any delayed take or prolonged neutropenia with this therapeutic regimen when compared to other bone marrow transplant patients, who did not receive this antimicrobial therapy. All patients tolerated this regimen well, adverse drug reactions did not occur.We conclude that teicoplanin is a potentially effective and safe antimicrobial agent in patients with life-threatening septicaemia after bone marrow transplantation and might be considered a useful, nontoxic agent treating infections not responding primarily to beta-lactams and aminoglycosides.

The ability to transfer new genetic information into hematopoietic cells provides a new and promising approach to address questions concerning stem cell commitment and proliferation. It should be possible, for instance, to introduce developmentally regulating genes, oncogenes, and genes encoding for growth factors into a number of different hematopoietic cells, thereby modulating the system in a precise way. Another aspect of this approach might be that certain human genetic defects may be corrected by the insertion of a functional gene into primitive bone marrow cells of the hematopoietic system.

Studies have shown that recombinant human alpha interferon (rIFN α) inhibits the growth of colonies of multipotential stem cells from human bone marrow. This report demonstrates that rIFN α inhibits the growth of such colonies from the bone marrow of patients with chronic myelogenous leukemia (CML) to a greater extent than from bone marrow of healthy individuals. It also shows that T lymphocyte colonies subcloned with interleukin 2 (IL-2) from CML mixed colonies were inhibited more by rIFN α than were similar colonies subcultured from normal mixed colonies. The report demonstrates that the Ph' chromosome is present in such T cell colonies subcultured from CML mixed colonies. When mixed colonies were grown from CML bone marrow in the presence of rIFN α, Ph' negative colonies were observed, whereas no such Ph' negative mixed colonies grew from a similar number of bone marrow cells incubated without rIFN α. These observations confirm that T lymphocytes derived from bone marrow stem cells are from the CML clone, and that the inhibition of growth of Ph' positive colonies, by rIFN α permits the growth of residual normal stem cells. The disappearance of the Ph-chromosome in subclones of T lymphocytes supports the notion of nonclonal hematopoiesis in patients with CML.

Chronic renal failure is often associated with severe anemia, subnormal levels of erythropoietin (EPO), and the presence of erythropoietic inhibitors. We studied an anephric patient with polycystic kidney disease maintaining a hemoglobin level of 13-14 g. Biochemical, endocrine, and hematological parameters were all within normal limits. EPO levels examined on two occasions during the clinical investigation were found to be low to subnormal. Plasma of this patient supported human bone marrow erythroid colony formation without the addition of exogenous EPO. Following reversed-phase high pressure liquid chromatography (HPLC) and gel filtration HPLC of the patient's plasma, a small polypeptide was identified that stimulated thymidine incorporation into fetal calf liver cells and supported erythroid colony formation of human marrow cells. This molecule is different from EPO with respect to its molecular weight and its functional and chromatographic properties. In this report we provide evidence of a human plasma-derived peptide of an anephric patient regulating late erythropoiesis.

In situ hybridization provides a powerful tool to detect specific mRNA sequences at the cellular level. We have applied a modified in situ hybridization technique using specifically prepared regular glass microscope slides to evaluate mRNA levels in cells of small samples. Cells were derived from in vitro colonies or isolated by fluorescence-activated cell sorting and deposited on the slides. These slides were coated with polysiloxane, sparing small circular areas where adherent cells attach and can be grown directly; after preincubation of the collection areas with fibronectin, the slides can also be used to deposit and to grow nonadherent cells. In situ hybridization was performed with 35S-labeled probes. Acetylation of the slides and the cells prior to hybridization, the addition of vanadyl-ribonucleoside complexes, and a prehybridization step were found to be necessary to optimize signal-to-noise ratios, as shown by evaluation of c-myc-specific mRNA in phytohemagglutinin-stimulated T4-lymphocytes. This technique might be very useful to study mRNA expression in small samples of hemopoietic cells.

Retroviral vectors containing the selectable bacterial gene for G418 resistance (neo) were used to demonstrate gene transfer into primary human bone-marrow progenitor cells. To obtain populations of cells in which a high proportion of cells were expressing the neo gene, several important modifications were made to earlier procedures. Cells from normal donors were infected in vitro, were exposed to high concentrations of G418 for two days in liquid culture to enrich for cells expressing the neo gene, and were plated in semisolid medium. Gene transfer and expression were detected in colonies arising from progenitors of granulocyte-macrophage and erythroid lineages. Survival curves indicated that a high proportion of progenitor cells, approaching 100%, were G418 resistant. Furthermore, addition of growth factors contained in 5637-conditioned medium to the bone marrow improved the recovery of G418-resistant progenitors twofold to threefold. In addition to these biological measurements of gene expression in progenitor cells, significant levels of neo-specific RNA, similar to the levels of RNA expression in the virus-producing fibroblast cell line, were detected in the bone marrow cells after preselection. These results demonstrate that retrovirus vectors can be used successfully to transfer genes at high efficiency into progenitor cells in the human blood-forming system.

Human hemopoietic progenitor cells were examined for the expression of glycoprotein IIIa (GPIIIa). This protein, which forms the beta-subunit of the GPIIb/IIIa receptor for cytoadhesive proteins as well as the beta-subunit of the vitronectin receptor, represents the most sensitive cell surface marker so far identified for the megakaryocytic lineage. Bone marrow cells were fractionated by a discontinuous Percoll gradient to separate cells that form megakaryocytic colonies in culture (1.05 greater than rho less than 1.077 g/ml). Density centrifugation was followed by indirect immunopanning to select for an enriched population of progenitor cells depleted of most of the mature cells of the myeloid, lymphoid, and erythroid lineages. This cell suspension was labeled with antibodies directed against determinants of GPIIIa and analyzed using a fluorescence-activated cell sorter (FACS). Fractions of cells were sorted and analyzed for the ability to form hemopoietic colonies in culture. Our study demonstrated that megakaryocytic progenitor cells (CFU-M) as well as granulocyte-macrophage colony-forming units (CFU-C), erythroid colony-forming units (BFU-E), and mixed lineage colony-forming units (CFU-GEMM) express HLA-DR antigens but lack GPIIIa. Therefore GPIIIa represents a marker that is not present on hemopoietic progenitor cells, but is expressed on the progenies of CFU-M. In view of the importance of GPIIIa as a component of receptors for cytoadhesive proteins, this finding may help to elucidate the adhesive interactions between early hemopoietic cells and bone marrow interstitium.

Chronic myelogenous leukemia (CML), polycythemia vera, essential thrombocythemia, primary myelofibrosis (PMF) and acute myelofibrosis (acute megakaryoblastic leukemia) form a group of hematological diseases, called myeloproliferative disorders, with varying patterns of lineage involvement in each disorder. There is laboratory and clinical evidence that bone marrow fibrosis in these diseases is promoted, at least in part, by platelet-derived growth factor (PDGF) (for review s. 1,2). PDGF displays growth promoting activity for stroma cells (for review 8.3–5) an¿ possibly, by indirect action for hemopoietic progenitor cells6–8. Its synthesis has been shown by a number of cells, including megakaryocytes, and the B-chain of PDGF is encoded by the c-sis protooncogene3–5.

Bone marrow cells from 10 marrow transplant donors were treated with an immunotoxin, which couples A-chain of ricin with a monoclonal anti-T-cell antibody T101 to prevent graft-versus-host disease by the elimination of mature T-cells. Marrow cells treated with the anti human T-cell immunotoxin (IT101) were cultured for erythropoietic colonies, granulocytic colonies, and multilineage hematopoietic colonies (CFU-GEMMT) containing myeloid cells and T-cells, and optimal conditions were defined for the elimination of T-cells present in the harvested donor marrow prior to marrow transplantation. Marrow samples purged with IT101 were examined for residual T-cells by fluorescence activated cell sorting, using anti-T-cell antibodies, [3H]-thymidine incorporation after PHA stimulation, and an assay for clonogenic T-cells. The number of T-cell colonies observed in the treated marrows was less than 5% of the number in comparable unpurged donor marrows. Treatment with IT101 did not alter the plating efficiency of hematopoietic colonies compared to untreated donor marrow cells. These data suggest that multilineage progenitors responsible for the reconstitution of the recipient hematopoietic system are not affected by marrow IT101 purging. The clinical data on 10 patients indicate that the depletion of T-cells in the donor marrow with IT101 is effective in decreasing the severity of acute graft-versus-host disease in allogeneic marrow transplantation and warrants continued investigation.

Chronic myelogenous leukemia (CML) is a myeloproliferative disorder in which the neoplastic transformation of a pluripotent stem cell results in the proliferation and accumulation of myeloid cells and their progenitors. Clinically, the disease is divided into three phases: a chronic phase of 3–4 years’ duration followed by an accelerated phase of 3–5 months, and then leading into an acute phase, i. e., blast crisis lasting 2–4 months [1]. During the chronic phase, the neoplastic clone represents the majority of the replicating myeloid cells. These cells and their progenitors normally respond to normal myelopoietic growth factors. In the acute phase, in contrast, the leukemic cells loose their ability to differentiate and mature normally. In CML a specific chromosome abnormality, the Philadelphia chromosome (Ph1) (22q-) is present in 90%–95% of patients [2]. The Ph1 chromosome results, in most instances, from a balanced reciprocal translocation between chromosome 9 and 22 [t(9;22)] [3].

Megakaryocytopoiesis represents one of several differentiation pathways that hematopoietic stem cells may enter. Cells representing intermediate stages of differentiation between pluripotent stem cells and maturing megakaryocytes are called megakaryocytic progenitor cells. They are identified in human bone marrow and peripheral blood by their ability to proliferate in culture (colony forming unit-megakaryocyte, CFU-M); at some point they lose the capacity for cell division and acquire the ability for endoreduplication of DNA, a phenomenon that is unique to the megakaryocyte lineage.

Effective hematopoiesis is a multistep phenomenon. It consists in the presence of pluripotent hematopoietic stem cells (HSC), their proliferation and self-maintenance, their differentiation into various committed lineages of specific progenitors, their orderly maturation into functional cells that are released into the circulation in an orderly fashion in response to the body's demand. Increasing numbers of hematopoietic factors are being purified to homogeneity and/or cloned. The availability of sufficient quantities of these regulators promises a new area for research into the physiology and pathophysiology of the hematopoietic system. The purpose of this overview is to consider some newly-developed concepts in the field of hematopoiesis, with regard to regulatory control mechanisms and cellular interactions.