Interferon-gamma modulates the lipopolysaccharide-induced expression of AP-1 and NF-kappa B at the mRNA and protein level in human monocytes.

Interferon-gamma (IFN-gamma) modulates the expression of several cytokines by human monocytes at the transcriptional level. In view of these findings, we analyzed the effects of IFN-gamma on the expression of different transcription factors in activated human monocytes. Priming of human monocytes with IFN-gamma resulted in the down regulation of c-fos and c-jun mRNA in response to stimulation with lipopolysaccharide (LPS) compared to the effects of LPS alone. Not only was this effect observed at the mRNA level, but activator protein-1 (AP-1) DNA binding capacity was affected as well, A strong reduction was observed in the LPS-induced DNA-binding activity of AP-1 in the presence of IFN-gamma. LPS-stimulated monocytes showed an increased expression of p105 mRNA, the precursor of the p50 subunit of the transcription factor nuclear factor-kappa B (NF-kappa B), while no effect was noticed on the expression of p65 mRNA. In contrast, IFN-gamma priming did not affect the expression of p105 transcripts but enhanced the expression of p65 mRNA (two-fold). Priming with IFN-gamma followed by LPS stimulation resulted in a further increase in the expression of p65 mRNA. This was due to an increase in the half-life of p65 mRNA (75 vs 150 minutes). Electrophoretic mobility shift assays (EMSAs) demonstrated that unstimulated monocytes predominantly expressed p50 NF-kappa B. Stimulation with LPS or IFN-gamma resulted in the expression of p50 and p65 subunits, while the combination of IFN-gamma plus LPS caused a further increase in the expression of NF-kappa B. With Western blotting, it was shown that nuclear extracts from monocytes contained p50 and p65 protein in response to LPS and IFN-gamma stimulation. However, the combined stimulation did not result in enhanced p50 and p65 protein expression. The effects of IFN-gamma on the transcription factors were specific, since no change was observed in the expression of NF-IL-6 or I kappa B alpha, the inhibitor of NF-kappa B. We conclude that the effects of IFN-gamma on the expression of the transcription factors AP-1 and NF-kappa B may be important for the modulatory effects of IFN-gamma on the cytokine expression in activated human monocytes.

In polycythemia vera human interleukin 3 and granulocyte-macrophage colony-stimulating factor enhance erythroid colony growth in the absence of erythropoietin.

To further define the growth factors required for the in vitro proliferation of erythroid progenitors in polycythemia vera (PV), we have compared the ability of interleukin 3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) to support the growth of erythropoietin (Epo)-dependent and -independent erythroid colony formation. By using nonadherent mononuclear cells from peripheral blood, Epo-dependent colony formation was enhanced by IL-3 and GM-CSF in PV patients. Comparable results were obtained with normal erythroid progenitors. Augmenting effects of IL-3 and GM-CSF were observed on spontaneous erythroid colony formation, i.e., erythroid colony formation in the absence of exogenous supplied Epo. This was not due to a small amount of Epo in the culture media because an anti-Epo antibody did not prevent endogenous colony formation, nor did it prevent the enhancing effects of IL-3. Finally it was observed that in contrast to IL-3, monocyte depletion was required for the enhancing effects of GM-CSF on erythroid colony formation. These results provide evidence that endogenous colony formation in PV is independent of Epo but can be augmented by IL-3 or GM-CSF.

Gold probe choice in simultaneous detection of human lymphocyte surface antigens at the ultrastructural level.

A double immunogold-labeling method in immunoelectron microscopy was used for simultaneous detection of two antigens by monoclonal antibodies [OKT 8 (CD 8), anti-Leu-7, anti-Leu-11b (CD 16)] on lymphocytes in suspension. The combination of gold probe size (5 nm and 15 nm) and monoclonal antibody was found to be decisive for detecting double-labeled cells with the OKT 8+, Leu-11b+ phenotype. The combinations of OKT 8 labeled with the 5-nm gold probe (OKT 8(5] and anti-Leu-11b with the 15-nm gold probe (Leu-11b15) gave double-labeled cells; the reverse situation, using OKT 8 with a 15-nm gold probe (OKT 8(15] and anti-Leu-11b with a 5-nm gold probe (Leu-11b5), did not. Double-labeled OKT 8+, Leu-7+ cells were detected irrespective of which gold probe combination was applied. Our findings indicate that although the double immunogold-labeling method is well suited for study of lymphocyte subsets, it is important to determine suitable combinations of gold probe sizes and monoclonal antibodies for the lymphocyte subset under study, taking into account surface antigen density, so that double labeling ensues.

Modulation of the intestinal microflora by the non-absorbable antibiotic vancomycin leads to a reduction of the tumor load in liver and spleen in a leukemic rat model.

Based on previous studies where it was shown that non-absorbable antibiotics can influence the normal hematopoiesis via changes in factors related to the intestinal microflora, the influence of vancomycin on the progression of acute myeloid leukemia was investigated in the BNML rat model. Oral vancomycin, which selectively reduces Gram-positive bacteria in the gut, leads to diminution of the leukemic load in liver and spleen by 30-60%. This 'antileukemic effect' is not dependent on Gram-negative bacteria as source for endotoxin. The presumed mechanism is a decrease of the leukemic growth fraction caused by alterations in the absorption of substances from intestinal Gram-positive bacteria.

A prospective, randomized, double-blinded, placebo-controlled trial of empirical teicoplanin in febrile neutropenia with persistent fever after imipenem monotherapy.

Glycopeptide antibiotics are used extensively in the empirical treatment of febrile patients with neutropenia. To come to a more rational and restricted application of these expensive drugs and to reduce the risk of emergence of resistance, we carried out a prospective, double-blinded, placebo-controlled single-centre study to investigate whether the addition of teicoplanin improved the outcome of neutropenic patients who remained febrile after 72-96 h of imipenem monotherapy. Patients with known infections caused by imipenem-resistant microorganisms were excluded. From the 114 evaluable episodes (out of a total of 125) in 105 patients who met the eligibility criteria, 56 episodes were randomized to receive teicoplanin and 58 to placebo. At 72 h after the start of the assigned intervention, 52 (45.6%) of the patients were afebrile; at the end of the aplastic phase, 10 (8.8%) had succumbed. There was no difference between the two study arms. When febrile episodes were subdivided between microbiologically documented infections, clinically documented infections and fevers of unknown origin, again no significant differences were observed. With the exception of methicillin-resistant bacteria, Gram-positive infections seemed to respond well to imipenem monotherapy. It is concluded that the addition of teicoplanin on empirical grounds, i.e. for persistent fever only, is not necessary and that the use of glycopeptides should be restricted to well-defined clinical situations where methicillin-resistant bacteria are involved. Furthermore, it seems that many neutropenic patients respond slowly over more than 72-96 h even when they are treated with antibacterial drugs such as imipenem that are effective against the causative microorganism.

Interferon-alpha induction of lymphocytes containing parallel tubular structures.

The induction by IFN-alpha in peripheral blood lymphocytes of parallel tubular structures (PTS) and/or electron-dense granules occurring in a minority of peripheral blood lymphocytes was examined. IFN reportedly augments natural killer (NK) cell activity of large granular lymphocytes (LGL); these cells contain PTS and/or electron-dense granules. Normal peripheral blood mononuclear cells were incubated with IFN-alpha and surface antigen expression was measured by means of indirect immunofluorescence and, at the ultrastructural level, using gold labelled monoclonal antibodies. Surface antigen reactivity with the monoclonal antibodies OKT 3, 4, 8 and Anti-Leu-7 (HNK-1) showed no difference between the IFN-alpha incubation and non-IFN-alpha groups. However, electron microscope investigation revealed significant absolute increases in the percentage of OKT 8+ and Anti-Leu-7+ cells which were PTS-positive after IFN-alpha treatment compared with the control groups. The cytotoxicity assay using the K562 cell line showed enhanced lytic activity. Our results suggest that cells coexpressing the OKT 8 and Leu-7 antigens may be responsible for a minor proportion of the increase in PTS but that IFN-alpha mainly induces PTS and/or associated structures in cells which express the OKT 8+ antigen. These PTS+/OKT 8+ cells may contribute to enhanced cell cytotoxicity.

Preparation and storage of "leukocyte-depleted single donor" platelet concentrate: a step forward in effective haemotherapy.

"Leukocyte-depleted" platelet concentrates (LD-SDPC) were prepared by cotton-wool and by polyester filtration of "leukocyte-poor" PC collected by the elutriation technique. The storage characteristics of LD-SDPC were comparable to those of the filtered pools of multiple donor platelet concentrates (LD-MDPC). However, LD-SDPC were less activated and less damaged than LD-MDPC over a 7-day storage period, as evidenced by beta-thromboglobin percent release and lactic dehydrogenase percent leakage in both products. LD-SDPC were prophylactically transfused to 12 thrombocytopenic patients; the mean bleeding time was shortened from 12 min 20 sec to 4 min 39 sec. Corrected count increment (CCI) was 22.8 at 1 hr and 14.1 at 24 hr compared to 15.5 and 10.0, respectively, with standard PCs.

Mast cell growth factor modulates CD36 antigen expression on erythroid progenitors from human bone marrow and peripheral blood associated with ongoing differentiation.

To study the differentiation process of erythroid progenitors from normal human bone marrow and peripheral blood, CD34/CD36 sorted cells were cultured in the presence of Erythropoietin (Epo) and Epo plus mast cell growth factor (MGF). The CD34+/CD36- cell fraction from bone marrow supported 74 +/- 33 erythroid burst forming units (BFU-E)/10(4) cells (mean +/- SD, n = 4) in the presence of Epo, which increased 2.1-fold by coculturing with MGF. However, erythroid colony-forming units (CFU-E) were not cultured from the CD34+/CD36- cell fraction. In contrast, the CD34-/CD36+ cell fraction supported CFU-Es in the presence of Epo (152 +/- 115/10(5)) or Epo plus MGF (180 +/- 112/10(5)), whereas BFU-Es were hardly noticed. However, the transition of the BFu-E to CFU-E was observed by incubating CD34+/CD36- cells (10(4)/100 microL) in suspension with Epo plus MGF for 7 days followed by Epo in the colony assay. This was reflected by the appearance of CD34-/CD36+/Glycophorin A+/CD14- cells. In addition high numbers of CFU-Es (1,000 +/- 150, n = 4) were cultured from this cell fraction. In contrast to bone marrow erythroid progenitors, no peripheral blood CFU-Es were cultured from either the CD36+ or CD36- fraction, whereas BFU-Es were predominantly present in the CD36+ fraction. However, the CD34+ progenitor cell from peripheral blood did have intrinsic capacity to differentiate to CFU-Es because CD34+/CD36- cells incubated with Epo plus MGF for 7 days and followed by Epo in the colony assay, supported high numbers of CFU-Es (1,200 +/- 400, n = 3). To study whether additional growth factors have similar effects on erythroid progenitors, experiments were performed with interleukin 1 (IL-1), IL-3, and IL-6. IL-1 and IL-6 did not modulate the Epo supported proliferation and differentiation. In contrast, IL-3 in the presence of Epo did support CFU-Es, from CD34+/CD36- cells after 7 days in suspension culture. However, flow cytometry analysis showed that Epo plus IL-3 not only supported CD34-/CD36+/Glycophorin A+ cells but also CD36+/CD14+ cells, indicating the differentiation along different cell lineages. In summary, the data show a phenotypic distinction between bone marrow and peripheral blood erythroid progenitors with regard to CD36 expression. In addition, the results suggest that Epo plus MGF or IL-3 and preincubation in suspension culture are prerequisites for the transition of the BFU-E to the CFU-E.

Improved outcome of adult acute lymphoblastic leukaemia by moderately intensified chemotherapy which includes a 'pre-induction' course for rapid tumour reduction: preliminary results on 66 patients.

Sixty-six consecutive adult patients with acute lymphoblastic leukaemia (ALL) were treated with intensified chemotherapy which included a 'pre-induction' course of cytarabine (AraC) and etoposide (VP16) when the white blood cell count (WBC) was > or = 30 x 10(9)/l (18 patients), and maintenance chemotherapy with regular intensifications for a total treatment duration of 3 years. Patients with a mediastinal mass (17) received consolidation courses with intermediate-dose AraC and VP16 followed by mediastinal irradiation. 11 patients underwent allogeneic bone marrow transplantation in first complete remission (CR). 58 patients (87.9%, CI 77.5-94.6) attained CR; with a median follow-up of 7 years, 35 of them (60.3%, CI 46.6-73.0) remain in CR. Toxicity was mild, although three patients died during remission induction, including two who were over 70 years of age. 23 patients (39.7%, CI 27.1-53.4) relapsed, seven of them primarily in the central nervous system (CNS), necessitating intensification of CNS-directed therapy. Only one of 13 patients with WBC 30-100 x 10(9)/l, but eight of nine with WBC > 100 x 10(9)/l, relapsed. The survival of older patients in CR did not differ from younger patients. The outcome of ALL in adult patients could thus be improved by slight intensification of treatment whilst keeping the toxicity within acceptable limits. 'Pre-induction' with AraC and VP16 might improve the prognosis, especially in patients with WBC < 100 x 10(9)/l. Patients with WBC > 100 x 10(9)/l, however, almost always relapse, and the intensified chemotherapy might not be tolerated well by patients over 70 years of age.

Risk of transmission of human immunodeficiency virus (HIV) by heat-treated factor VIII concentrates in patients with severe hemophilia A.

At the change from unheated to heat-treated Factor VIII concentrates for the treatment of hemophilia A, 17 severe adult hemophiliacs (mean monthly dose, 4927 IU) were evaluated prospectively for signs of infection with human immunodeficiency virus (HIV). Viral serology and lymphocyte subpopulations (OKT3, OKT4, and OKT8-positive cells) were examined monthly for 1 year. One patient seroconverted for HIV in the enzyme-linked immunoabsorbent assay but was positive on the Western blot analysis from the outset. There was a slight but significant increase in OKT4+ cells and OKT4/OKT8 ratio. These data suggest that heat-treated Factor VIII concentrates even when used in large amounts have a low risk of transmitting HIV.