Interferon gamma selectively inhibits very primitive CD342+CD38- and not more mature CD34+CD38+ human hematopoietic progenitor cells.

To assess the effects of interferon gamma (IFN-gamma) on very primitive hematopoietic progenitor cells, CD34(2+)CD38- human bone marrow cells were isolated and cultured in a two-stage culture system, consisting of a primary liquid culture phase followed by a secondary semisolid colony assay. CD34(2+)CD38- cells needed at least the presence of interleukin 3 (IL-3) and kit ligand (KL) together with either IL-1, IL-6, or granulocyte-colony-stimulating factor (G-CSF) in the primary liquid phase in order to proliferate and differentiate into secondary colony-forming cells (CFC). Addition of IFN-gamma to the primary liquid cultures inhibited cell proliferation and generation of secondary CFC in a dose-dependent way. This was a direct effect since it was also seen in primary single cell cultures of CD34(2+)CD38- cells. The proliferation of more mature CD34+CD38+ cells, however, was not inhibited by IFN-gamma, demonstrating for the first time that IFN-gamma is a specific and direct hematopoietic stem cell inhibitor. IFN-gamma, moreover, preserves the viability of CD34(2+)CD38- cells in the absence of other cytokines. IFN-gamma could, therefore, play a role in the protection of the stem cell compartment from exhaustion in situations of hematopoietic stress and may be useful as stem cell protecting agent against chemotherapy for cancer.

Pentostatin in refractory chronic lymphocytic leukemia: a phase II trial of the European Organization for Research and Treatment of Cancer.

Pentostatin was used to treat 26 patients with advanced B-cell chronic lymphocytic leukemia resistant to conventional treatment. Twenty patients had progressive disease on previous regimens and six had had partial remission and then relapsed 3-34 months after previous chemotherapy. Eleven patients had previously been treated with three different regimens. 10 had been treated with two regimens, and five had been treated with one regimen. Pentostatin was administered at a dosage of 4 mg/m2 weekly for 3 weeks, then 4 mg/m2 every other week for 6 weeks and once a month for 6 months. Seven of 26 assessable patients (27%) achieved partial remission and five (19%) achieved clinical improvement. The median duration of partial remission until relapse or death was 210 days. Myelosuppression was minor and transient in responsive patients, indicating some degree of selective effect on lymphocytes. Except for one patient who died of cerebral hemorrhage during the first 6 weeks of treatment, no drug-related deaths were registered. Major toxic effects included nausea in 17 patients (mainly grade 1), infections in 15, and liver enzyme elevations in five. Thus, pentostatin is active, even in patients with advanced B-cell chronic lymphocytic leukemia that is refractory to multiple chemotherapy regimens. Response can be achieved with mild myelosuppression.

Morphological evidence for a motile behavior by plasma cells.

Immunofluorescence microscopy of the adherent layer of human long-term bone marrow cultures (HLTBMCs) consistently revealed motility-associated features in a part of the plasma cells. Not only were small surface blebs observed, but also long extensions whose morphology was reminiscent of neuronal axons. The observations were made in all HLTBMCs established with normal as well as myelomatous bone marrow (BM). Plasma cells with long extensions were also noticed on May-Grünwald-Giemsa smears of original uncultured BM from normal individuals and patients with myelomatous disorders. This suggests that the motility-associated morphological features occur in vivo as well as in vitro. The observations were most striking in the one case of plasma cell leukemia, both in culture as well as in the BM smear.

Effects of interleukin-4 (IL4) on myelopoiesis: studies on highly purified CD34+ hematopoietic progenitor cells.

We studied the effects of interleukin 4 (IL4) on myelopoiesis supported by either granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage CSF (GM-CSF) or IL3 on purified CD34+ bone marrow progenitor cells. IL4 stimulates the colony-forming unit granulocyte (CFU-G) induced by G-CSF and inhibits all colony types supported by either IL3 and GM-CSF, although inhibition of CFU-M (macrophage) was significantly stronger than that of CFU-G and CFU-GM. When the cells were first incubated in liquid culture for 4 days in IL4, followed by agar culture in G-CSF, there was a significant increase in the number of CFU-G compared to cells which had been incubated in medium alone for 4 days before plating out in agar containing G-CSF. The inhibitory effects of IL4 on GM-CSF or IL3 supported colony formation, however, disappear with sequential incubation in IL4 in liquid culture followed by culture in agar with either GM-CSF or IL3.

The nature of the adherent hemopoietic cells in human long-term bone marrow cultures (HLTBMCs): presence of lymphocytes and plasma cells next to the myelomonocytic population.

An immunological analysis of the nonmacrophage hemopoietic cells in the adherent layer of human long-term bone marrow cultures was performed in situ. It revealed not only the expected granulocytic and monocytic cells, but an important lymphoid population as well. The latter consisted of cells bearing the markers of mature T lymphocytes, B lymphocytes, and plasma cells. These cells were also present in the hemopoietic foci of the adherent layer, the so-called cobblestone areas. Although more CD8+ than CD4+ lymphocytes were generally present in the initial bone marrow inoculum, both the adherent layer and the nonadherent fraction disclosed a preponderance of CD4+ cells after a short period of culture. The majority of the lymphoid cells were present in the adherent layer, rather than in the nonadherent fraction of the cultures. The long-term presence of lymphoid elements in the adherent layer suggests their affinity for the bone marrow stroma and the possible enhancement of their persistence in vitro by contact with these cells.

Human long-term bone marrow cultures (HLTBMCs) in myelomatous disorders.

Human long-term bone marrow cultures (HLTBMCs) were established with bone marrow (BM) collected from five patients with myelomatous disorders (four with multiple myeloma, one with plasma cell leukemia). In all cases, up to at least 6 weeks of culture, there was a persistence of the monoclonal plasma cell population in the adherent layer of the culture. In some cultures proliferating plasma cells could be demonstrated by the Ki-67 monoclonal antibody. In all instances a paraprotein could be shown in the conditioned medium. This study demonstrates that malignant plasma cells, in analogy to their normal counterparts, have an affinity for the BM stroma and suggests that their long-term survival might be enhanced by their interaction with it.

Karyotypic evidence for the leukemic involvement of the erythroblasts in erythroleukemia.

With the use of a monoclonal anti-glycophorin A antibody and flow cytometric cell sorting, an erythroleukemic bone marrow sample was separated in highly purified erythroblast and myeloblast fractions. Similar karyotypic anomalies were found in both cell populations as in the unseparated bone marrow. This study confirms that acute nonlymphocytic leukemia can originate at the level of a multipotential hemopoietic stem cell.

A quantitative and dynamic study of endothelial cells and megakaryocytes in human long-term bone marrow cultures.

The quantitative evolution of endothelial cells (ECs) in Dexter-type human long-term bone marrow cultures (HLTBMCs) was investigated. Using monoclonal antibodies directed against von Willebrand factor (vWF) and against membrane antigens (EN-4 and PAL-E), a low percentage--usually less than 1% of stromal cells--of ECs was detected in all confluent cultures established from 11 different bone marrow samples. Generally these cells are not associated directly with the areas of myelopoiesis ("cobblestone areas"). ECs cannot be demonstrated in the adherent layer of most young, non-confluent, and of some old, HLTBMCs. In some instances, morphological features suggestive of dynamic behavior were seen (sprouting, canal formation). In addition, a very low proportion of vWF-positive megakaryocytic cells was found in 4 of 11 cultures, always in direct contact with the stromal fibroblastic cells.

Stromal populations and fibrosis in human long-term bone marrow cultures.

An immunofluorescence study of the adherent layer of human long-term bone marrow cultures (HLTBMC) revealed the following surface markers on the different stromal cell populations: stromal fibroblastic cells CD10+, FIB86.3+, CD13+, CD71+; adipocytes CD10+, FIB86.3-, CD13+, CD71-/+; and macrophages CD10-/+, FIB86.3+, CD13+, CD71-/+, CD14+, CD33+, CD25+, HLA-DR+, CD4+, CD19+, CD45+. The markers of the stromal fibroblastic cells in HLTBMC were similar to those of twice-passaged fibroblasts not only from bone marrow and spleen, but also from a hemopoietic non-supportive organ such as the skin. Some of the cultured human umbilical vein endothelial cells used as controls were found to be CD25+, demonstrating for the first time the interleukin-2 receptor p55 chain on normal non-hemopoietic cells. The stromal fibroblastic cells are overrepresented compared to the small non-macrophage hemopoietic cell population in the adherent layer of HLTBMC. In addition, silver staining revealed an increased reticulin content in most of the HLTBMC. An excessive growth of stromal fibroblastic cells and an excessive deposition of their product, the reticulin fibers, are the hallmark of myelofibrosis. The finding of equivalent observations in HLTBMC suggests that the hitherto unexplained, premature quenching of hemopoiesis in HLTBMC might at least partly be due to mechanisms similar to those operating in myelofibrosis in vivo.

Interleukin 4 and interferon gamma costimulate the expansion of early human myeloid colony-forming cells. Proposal of a model for the regulation of myelopoiesis by interleukin 4 and interferon gamma and its integration with the regulation of the immune response.

We have previously shown that interleukin 4 (IL-4) and interferon gamma (INF-gamma) reciprocally regulate the production of granulocytes and monocytes from mature monopotential hematopoietic progenitor cells, while at the level of the very primitive stem cells IFN-gamma is a selective inhibitor of proliferation and differentiation, and IL-4 has weak stimulatory effects. We investigated the effects of IL-4 and IFN-gamma on the expansion in suspension culture of myeloid colony-forming cells (CFCs) induced by either IL-3 or IL-1+IL-3, using on the one hand more differentiated CD34+HLA-DR strongly positive (HLA-DR++) and on the other hand more primitive Cd34+HLA-DR weakly positive (HLA-DR+/-) human bone marrow cells. It is shown that both IL-4 and IFN-gamma stimulate the IL-3- and IL-3+IL-1-induced expansion of the number of CFCs in the HLA-DR+/- population. In the presence, but not in the absence of IL-1, additive effects of IL-4 and IFN-gamma were seen. We could not demonstrate any IL-3-like effect by IL-4 on early human hematopoietic progenitors. No expansion of CFC number was seen in the HLA-DR++ population. Based on these data and on data which we have published previously, a model for the regulation of myelopoiesis by IL-4 and IFN-gamma is proposed. In this model, IL-4 and IFN-gamma, which are both immune recognition induced inflammatory cytokines, both stimulate the expansion and recruitment of early myeloid progenitors, whereas at the level of their terminal differentiation, the balance between both cytokines determines whether preferentially monocytes/macrophages (IFN-gamma) or granulocytes (IL-4) are being produced. At the level of the most primitive cells, the inhibitory action of IFN-gamma might prevent differentiative exhaustion of the stem cell compartment in situations of hematopoietic stress.

Comparison of human long-term bone marrow cultures (HLTBMCs) established from fresh and post-cryopreservation bone marrow samples from living and cadaver donors.

Human long-term bone marrow cultures (HLTBMCs) were established from thawed post-cryopreservation, as well as from cadaver donor bone marrow (BM) samples. The longevity was similar in the different series of HLTBMCs examined. CFU-GM could be cultured out of cadaver donor BM. This indicates that previously healthy people, under the conditions generally accepted as suitable for organ donation, could become suitable donors for allogeneic BM transplantation.

Purpura fulminans in pneumococcal sepsis.

Two cases of pneumococcal sepsis in splenectomized patients were complicated by purpura fulminans. In addition, acute renal failure developed in both patients, and myolysis in one. Immunological findings in the patient with myolysis suggest a possible role of pneumococcal antigen-containing circulating immune complexes in the pathogenesis of these complications.

Differential regulation of the expression of CD38 and human leukocyte antigen-DR on CD34+ hematopoietic progenitor cells by interleukin-4 and interferon-gamma.

We studied the effects of interleukin-4 (IL-4) and interferon-gamma (IFN-gamma) on the expression of CD38 and human leukocyte antigen (HLA)-DR on purified CD34+ bone marrow progenitor cells. CD34+CD38- and CD34+HLA-DR- cells are largely nonoverlapping populations. After culture for 4 days in IFN-gamma, the expression of CD38 and HLA-DR is significantly increased and the disappearance of the CD38- and HLA-DR- populations is virtually complete. Moreover, IFN-gamma induces a population of CD34+ cells with a very high expression of CD38 (CD34+CD38++ cells), which were absent in the initial CD34+ population. IL-4 has no effect on the expression of CD38, but induces a limited but significant increase in the expression of HLA-Dr. After culture in IFN-gamma, CD34+ cells show a higher cloning efficiency of the colony-forming unit-macrophage (CFU-M) and burst-forming unit-erythroid (BFU-E) compared to cells cultured in medium alone. After culture in IL-4, a limited increase in CFU-granulocyte (CFU-G) and BFU-E is seen, whereas CFU-G, CFU-M, and BFU-E are increased after culture in IL-4 plus IFN-gamma. We further investigated the functional properties of the CD34+CD38++ cells generated in the presence of IFN-gamma. This cell population is highly enriched for BFU-E but partially depleted of CFU-M. Most of the CFU-M were found in the CD34+CD38+/-(CD34+CD38- and CD34+CD38+ cells) population.

Effects of interleukin-4 on myelopoiesis: localization of the action of IL-4 in the CD34+HLA-DR++ subset and distinction between direct and indirect effects of IL-4.

We compared the myelopoietic effects of interleukin-4 (IL-4) on CD34+HLA-DR+ and on CD34+HLA-DR++ bone marrow progenitor cells stimulated by either granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3). IL-4 stimulates G-CSF-induced colony-forming unit-granulocyte (CFU-G) and inhibits all colony types induced by GM-CSF and IL-3 in the HLA-DR++ population, but not in the HLA-DR+ population. In CD34+HLA-DR+ cells, however, a stimulation of G-CSF-supported CFU-G was also seen with sequential application of IL-4 in liquid cultures followed by G-CSF in agar cultures. In order to confirm that these are direct effects of IL-4, single-cell culture experiments were performed with CD34+HLA-DR++ cells. In these cultures IL-4 stimulates G-CSF-induced CFU-G and only inhibits colony-forming unit-macrophage (CFU-M) regardless of the CSF used to generate them.

Transforming growth factor-beta regulates the cell cycle status of interleukin-3 (IL-3) plus IL-1, stem cell factor, or IL-6 stimulated CD34+ human hematopoietic progenitor cells through different cell kinetic mechanisms depending on the applied stimulus.

The immediate cell kinetic response of highly purified human bone marrow progenitor cells (CD34+ sorted fraction) to the inhibitory effects of transforming growth factor-beta (TGF-beta) was studied using the BrdU-Hoechst flow-cytometric technique. The progenitor cells were stimulated with either interleukin-3 (IL-3) alone or with IL-3 in combination with IL-1, stem cell factor (SCF), or IL-6, and the inhibitory action of TGF-beta was evaluated in each phase of the first three consecutive cell cycles. Semisolid methylcellulose cultures were also performed to compare these initial events to the effects observed after 7, 14, and 21 days of incubation. Within the CD34+ compartment, the progenitor cells can be discriminated on a functional basis, i.e., in terms of TGF-beta sensitivity. Very primitive progenitors, recruited out of the G0 phase by IL-3 plus an early-acting factor (IL-1, SCF) are, upon addition of TGF-beta, arrested specifically in the G1 phase of the second cell cycle. In the clonogenic assays, the increased colony formation due to IL-1 or SCF was completely abolished by the counteracting effect of TGF-beta that diminished colony output back to the level of TGF-beta-plus-IL-3 supplemented colony growth. Addition of TGF-beta to CD34+ progenitors responding to IL-3 alone resulted in an overall retardation, but without an apparent specific accumulation of cells in any of the cell cycles. Finally, within the CD34+ compartment, there exists a subset of IL-3-responsive, but TGF-beta-insensitive, progenitor cells that were, upon addition of TGF-beta, not arrested at all. In conclusion, our results demonstrate that TGF-beta exerts different cell kinetic effects on CD34+ progenitor cell growth depending on the applied stimulus.

DNA fingerprints revealing common and divergent human DNA methylation patterns.

We compared DNA fingerprints of different cell populations from the same individuals, after separate digestion with the isoschizomers MboI and Sau3A. Methylation differences were observed within every individual when comparing fingerprints of Sau3A- with MboI-digested DNA, and of Sau3A-digested sperm with somatic DNA. In some cases, differences were also detected between fingerprints of Sau3A-digested somatic DNA originating from various cell sources. Methylation patterns common to all cell populations examined, including the germline, were observed with a higher frequency than divergent ones. These 'common methylations' are most likely to find their origin during early embryogenesis.

Assessment of anti-HLA antibodies in sera being tested for platelet reactivity by a platelet lymphocyte immunofluorescence test (PLIFT).

An indirect immunofluorescence test using a platelet mononuclear cell suspension is described. The test is a simple and sensitive method to assess the contamination by anti-HLA antibodies of sera being tested for platelet reactivity and eliminates the need to support two independent test systems such as lymphocytotoxicity and immunofluorescence testing. The test could be of value in routine platelet serology testing and platelet crossmatching.

Practical immunoevaluation in anesthesia.

Postoperative infection and cancer metastasis suppose weakened immunological resistance. Immunoevaluation is difficult for technical reasons and for individual variability. Proposed tests are hematology, cytochemistry, immunoglobulin and complement assays, membrane and cytoplasmic fluorescence, rosettes, lymphocyte transformation tests, skin tests, tests for granulocyte and monocyte function. Material and methods should be well standardized. Frozen serum and lymphocytes are peremptory for cellular and humoral evaluation during the different phases of anesthesia.

[Sulfasalazine allergy: fever, skin rash, hepatitis and T-lymphocytes]. / Sulfasalazine-allergie: koorts, huidrash, hepatitis en T-lymfocytose.

We describe a case of severe sulfasalazine allergy. Exacerbation of the symptoms occurred after unintentional rechallenge with co-trimoxazole, indicating that the reaction was triggered by the sulphonamide component. The clinical picture consisted of generalised adenopathy, hepatitis, high fever and a maculopapular skin rash. A bone marrow biopsy and skin biopsy both showed noncaseating granulomas. The white blood cell count rose to 90.10(9)/l with 40% atypical lymphocytes (plasmacytoid). They were identified by flow cytometry as activated T lymphocytes.