Importance of information following myocardial infarction: a study of the self-perceived information needs of patients and their spouse/partner compared with the perceptions of nursing staff

Time course studies revealed that the combination of Flt-3 ligand (FL), Steel factor (SF) and interleukin-3 (IL-3) did not elicit as large an amplification of the long-term culture-initiating cell (LTC-IC) population in serum-free cultures of CD34+ CD38- cord blood (CB) cells as was obtained in similar cultures of adult human CD34+ CD38- bone marrow (BM) cells (4- v 90-fold maximum increases), even though both total and colony-forming cell (CFC) numbers initially increased more rapidly in CB cultures. Multifactorial analysis of the short-term (10 d) effects of different cytokines identified FL and IL-6 in combination with the soluble IL-6 receptor (sIL-6R) as most important for expanding the CB LTC-IC population. In contrast, their counterparts in adult BM were most effectively stimulated by FL, SF and IL-3. For rapid generation of increased numbers of CFC, SF with either FL or IL-6/sIL-6R were found to be the most important contributors in cultures of CD34+ CD38- CB cells, whereas, in analogous BM cultures, IL-6/sIL-6R and TPO (in addition to FL, SF and IL-3) were required. These findings reinforce the principle of altered cytokine responsiveness as a hallmark of early haemopoietic cell differentiation and demonstrate how cytokine requirements may change during human ontogeny. Identification of conditions for optimizing the expansion of different subsets of primitive CB cells has additional important implications for clinical transplantation and gene transfer.     

Ex vivo culture of CD34+/Lin-/DR- cells in stroma-derived soluble factors, interleukin-3, and macrophage inflammatory protein-1alpha maintains not only myeloid but also lymphoid progenitors in a novel switch culture assay

We have demonstrated that long-term culture initiating cells (LTC-IC) are maintained in a stroma noncontact (SNC) culture where progenitors are separated from stroma by a microporous membrane and LTC-IC can proliferate if the culture is supplemented with interleukin-3 (IL-3) and macrophage inflammatory protein-1alpha (MIP-1alpha). We hypothesize that the same conditions, which result in LTC-IC proliferation, may also maintain lymphoid progenitors. Natural killer (NK) cells are of lymphoid lineage and a stromal-based culture can induce CD34+/Lin-/DR- cells to differentiate along the NK cell lineage. We developed a three-step switch culture assay that was required to demonstrate the persistence of NK progenitors in CD34+/Lin-/DR- cells assayed in SNC cultures supplemented with IL-3 and MIP-1alpha. When CD34+/Lin-/DR- progeny from the SNC culture were plated sequentially into "NK cell progenitor switch" conditions (contact with stromal ligands, hydrocortisone-containing long-term culture medium, IL-2, IL-7, and stem cell factor [SCF]) followed by "NK cell differentiation" conditions (contact with stromal ligands, human serum, no hydrocortisone, and IL-2), significant numbers of CD56+/CD3- NK resulted, which exhibited cytotoxic activity against K562 targets. All steps are required because a switch from SNC cultures with IL-3 and MIP-1alpha directly to "NK cell differentiation" conditions failed to yield NK cells suggesting that critical step(s) in lymphoid commitment were missing. Additional experiments showed that CD34+/CD33- cells present after SNC cultures with IL-3 and MIP-1alpha, which contained up to 30% LTC-IC, are capable of NK outgrowth using the three-step switch culture. Limiting dilution analysis from these experiments showed a cloning frequency within the cultured CD34+/CD33- population similar to fresh sorted CD34+/Lin-/DR- cells. However, after addition of FLT-3 ligand, the frequency of primitive progenitors able to develop along the NK lineage increased 10-fold. In conclusion, culture of primitive adult marrow progenitors ex vivo in stroma-derived soluble factors, MIP-1alpha, and IL-3 maintains both very primitive myeloid (LTC-IC) and lymphoid (NK) progenitors and suggests that these conditions may support expansion of human hematopoietic stem cells. Addition of FLT-3 ligand to IL-2, IL-7 SCF, and stromal factors are important in early stages of NK development.     

Efficient retroviral-mediated gene transfer to human cord blood stem cells with in vivo repopulating potential

Recent studies have shown efficient gene transfer to primitive progenitors in human cord blood (CB) when the cells are incubated in retrovirus-containing supernatants on fibronectin-coated dishes. We have now used this approach to achieve efficient gene transfer to human CB cells with the capacity to regenerate lymphoid and myeloid progeny in nonobese diabetic (NOD)/severe combined immunodeficiency (SCID) mice. CD34(+) cell-enriched populations were first cultured for 3 days in serum-free medium containing interleukin-3 (IL-3), IL-6, granulocyte colony-stimulating factor, Flt3-ligand, and Steel factor followed by two 24-hour incubations with a MSCV-NEO virus-containing medium obtained under either serum-free or serum-replete conditions. The presence of serum during the latter 2 days made no consistent difference to the total number of cells, colony-forming cells (CFC), or long-term culture-initiating cells (LTC-IC) recovered at the end of the 5-day culture period, and the cells infected under either condition regenerated similar numbers of human CD34(+) (myeloid) CFC and human CD19(+) (B lymphoid) cells for up to 20 weeks in NOD/SCID recipients. However, the presence of serum increased the viral titer in the producer cell-conditioned medium and this was correlated with a twofold to threefold higher efficiency of gene transfer to all progenitor types. With the higher titer viral supernatant, 17% +/- 3% and 17% +/- 8%, G418-resistant in vivo repopulating cells and LTC-IC were obtained. As expected, the proportion of NEO + repopulating cells determined by polymerase chain reaction analysis of in vivo generated CFC was even higher (32% +/- 10%). There was no correlation between the frequency of gene transfer to LTC-IC and colony-forming unit-granulocyte-macrophage (CFU-GM), or to NOD/SCID repopulating cells and CFU-GM (r2 = 0.16 and 0.17, respectively), whereas values for LTC-IC and NOD/SCID repopulating cells were highly and significantly correlated (r2 = 0.85). These findings provide further evidence of a close relationship between human LTC-IC and NOD/SCID repopulating cells (assessed using a >/= 6-week CFC output endpoint) and indicate the predictive value of gene transfer measurements to such LTC-IC for the design of clinical gene therapy protocols.     

Structurally specific heparan sulfates support primitive human hematopoiesis by formation of a multimolecular stem cell niche

Stem cell localization, conservation, and differentiation is believed to occur in niches in the marrow stromal microenvironment. Our recent observation that long-term in vitro human hematopoiesis requires a stromal heparan sulfate proteoglycan (HSPG) led us to hypothesize that such HSPG may orchestrate the formation of the stem cell niche. We compared the structure and function of HS from M2-10B4, a hematopoiesis-supportive cell line, with HS from a nonsupportive cell line, FHS-173-We. Long-term culture-initiating cell (LTC-IC) maintenance was enhanced by PG from supportive cells but not by PG from nonsupportive cells (P <.005). The supportive HS were significantly larger and more highly sulfated than the nonsupportive HS. Specifically, supportive HS contained higher 6-O-sulfation on the glucosamine residues. In agreement with these observations, purified 6-O-sulfated heparin and highly 6-O-sulfated bovine kidney HS similarly maintained LTC-IC. In contrast, completely desulfated heparin, N-sulfated heparin, and unmodified heparin did not support LTC-IC maintenance. Moreover, the supportive HS promoted LTC-IC maintenance but not differentiation of CD34(+)/HLA-DR- cells into colony-forming cells (CFCs) and mature blood cells. The supportive HS but not the nonsupportive HS bound both cytokines and matrix components critical for hematopoiesis, including interleukin-3 (IL-3), macrophage inflammatory protein-1 (MIP-1), and thrombospondin (TSP). Significantly more CD34(+) cells adhered directly to immobilized O-sulfated heparin than to N-sulfated or desulfated heparin. Thus, hematopoiesis-supportive stromal HSPG possessing large, highly 6-O-sulfated HS mediate the juxtaposition of hematopoietic progenitors with stromal cells, specific growth-promoting (IL-3) and growth-inhibitory (MIP-1 and platelet factor 4 [PF4]) cytokines, and extracellular matrix (ECM) proteins such as TSP. We conclude that the structural specificity of stromal HSPG that determines the selective colocalization of cytokines and ECM components leads to the formation of discrete niches, thereby orchestrating the controlled growth and differentiation of stem cells. These findings may have important implications for ex vivo expansion of and gene transfer into primitive hematopoietic progenitors.     

CD14+ cells in granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood mononuclear cells induce secretion of interleukin-6 and G-CSF by marrow stroma

The ability of granulocyte colony-stimulating factor (G-CSF)-mobilized peripheral blood mononuclear cells (G-PBMCs) to induce secretion of cytokines in primary long-term marrow cultures (LTC) or in the human marrow stromal cell line HS23 was compared with that of marrow mononuclear cells. Equal numbers of G-PBMCs or marrow mononuclear cells were added to stromal cultures, supernatants were harvested at day 4 and levels of interleukin-1 alpha (IL-1 alpha), IL-1 beta, IL-2, IL-6, G-CSF, and tumor necrosis factor alpha (TNF alpha) were determined. G-PBMCs induced 21.4-fold higher levels of IL-6 and 12.5-fold higher levels of G-CSF in LTC cocultures compared with marrow mononuclear cells and induced 20.6-fold more IL-6 and 6.3-fold more G-CSF when added to HS23 cells. Experiments using sorted populations of CD20+, CD3+, and CD14+ cells showed that CD14+ cells within G-PBMCs were responsible for triggering the production of IL-6 and G-CSF. The effect did not require cell-cell contact and was inhibited when neutralizing antibodies to IL-1 alpha and IL-1 beta were used in combination. In these experiments, the greater stimulating ability of G-PBMCs is most likely attributable to the greater number of CD14+ cells in G-PBMCs (26.1+% +/- 2.3%) compared with marrow (2.5% +/- 0.8%), because equal numbers of CD14+ cells sorted from marrow and G-PBMCs showed comparable ability to induce IL-6 and G-CSF when placed directly on stromal cells.     

Microclimate-dependent survival of unfed adult Ixodes scapularis (Acari:Ixodidae) in nature: life cycle and study design implications

We report that acetyl-N-Ser-Asp-Lys-Pro (AcSDKP), which removes progenitor cells from cell cycle, in combination with granulocyte-colony stimulating factor (G-CSF) can significantly improve myelorestoration following irradiation (7 Gy). Peripheral blood, spleen and bone marrow (BM) cell recovery and progenitor cell reconstitution [IL-3-responsive colony-forming cells (CFC) and high proliferative potential colony-forming cells (HPP-CFC)] were studied. Studies on the optimal schedule of AcSDKP administration revealed maximal effects on progenitor cells when AcSDKP was administered as a continuous infusion for 3 d starting 24 h prior to irradiation and used in combination with G-CSF. The numbers of CFC and HPP-CFC in the BM were significantly increased following irradiation in mice receiving AcSDKP and G-CSF as compared to either drug alone. The numbers of CFC in the spleen were significantly increased in mice receiving AcSDKP and G-CSF on days 10 and 14 as compared to AcSDKP alone, but not G-CSF. Similarly, CFC and HPP-CFC in the spleen were significantly increased in mice receiving AcSDKP and G-CSF on day 18 as compared to mice receiving PBS and G-CSF. These studies suggest that AcSDKP in combination with G-CSF may have potential for the protection of progenitor cells in patients undergoing intensive chemo- and/or radiotherapy.     

Stromal factors support the expansion of the whole hemopoietic spectrum from bone marrow CD34+DR- cells and of some hemopoietic subsets from CD34+ and CD34+DR+ cells

Ex vivo expanded bone marrow CD34+DR- cells could offer a graft devoid of malignant cells able to promptly reconstitute hemopoiesis after transplant. We investigated the specific expansion requirements of this subpopulation compared to the more mature CD34+ and CD34+DR+ populations. The role of stromal factors was assessed by comparing the expansion obtained when the cells were cultured in (1) long-term bone marrow culture (LTBMC) medium conditioned by an irradiated human BM stroma (CM), (2) medium supplemented with 15% FBS (FBSM) and (3) non-conditioned LTBMC medium (LTM) for 21 days. The effect of the addition of G-CSF (G) and/or of MIP-1alpha (M) to a combination of IL-3, SCF, IL-6 and IL-11 (3, S, 6, 11) was analyzed. Compared to CD34+DR- cells, CD34+ and CD34+DR+ cells gave rise to a similar number of viable cells and to a lower progenitor expansion. The expansion potential of CD34+ and CD34+DR+ cells was equivalent in CM and in FBSM except for both the emergence of CD61 + megakaryocytic cells and LTC-IC maintenance which were improved by culture in CM. In contrast, expansion from CD34+DR- cells was enhanced by CM for all the parameters tested. Compared to FBSM, CM induced a higher level of CFU-GM and BFU-E expansion and allowed the emergence of CD61+ cells. HPP-CFC were maintained or expanded in CM but decreased in FBSM. Compared to input, the number of LTC-IC remaining after 21 days of CD34+DR- expansion culture was strongly decreased in FBSM and variably maintained or expanded in CM. Comparison with LTM indicated that stroma conditioning is responsible for this effect. G-CSF significantly improved CFU-GM and HPP-CFC expansion from CD34+DR- cells without being detrimental to the LTC-IC pool. The growth of CD61+ cells was significantly enhanced by G-CSF in CM. Addition of MIP-1alpha had no significant effect either on progenitor expansion or on LTC-IC, regardless of culture medium. We conclude that factors present in stroma- conditioned medium are necessary to support the expansion of the whole spectrum of hematopoietic cells from CD34+DR- cells and to support the expansion of cell subsets from CD34+ and CD34+DR+.     

Mobilization and homing of peripheral blood progenitors is related to reversible downregulation of alpha4 beta1 integrin expression and function

Despite the wide use of mobilized peripheral blood (PB) progenitor cells (PBPC) for clinical transplantation the mechanism(s) underlying their mobilization and subsequent engraftment are still unknown. We compared the adhesive phenotype of CD34(+) colony-forming cells (CFC) in bone marrow (BM) and PB of normal donors before and after administration of granulocyte colony-stimulating factor (G-CSF) for 5 d. G-CSF-mobilized PB CFC cells adhered significantly less to BM stroma, fibronectin, and to the alpha4 beta1 binding fibronectin peptide, CS1, because of decreased expression of the alpha4 integrin. Since incubation of BM CD34(+) cells for 4 d with G-CSF at concentrations found in serum of G-CSF- treated individuals did not affect alpha4-dependent adhesion, G-CSF may not be directly responsible for the decreased alpha4-mediated adhesion of PB CFC. Culture of G-CSF-mobilized PB CD34(+) cells with cytokines at concentrations found in BM stromal cultures upregulated alpha4 expression and restored adhesion of mobilized PB CFC to stroma, fibronectin, and CS1. Adhesion of cultured, mobilized PB CFC to stroma and CS1 could not be further upregulated by the beta1 activating antibody, 8A2. This indicates acquisition of a maximally activated alpha4 beta1 integrin once PB CFC have been removed from the in vivo mobilizing milieu. Thus, decreased alpha4 expression on CD34(+) CFC in PB may be responsible for the aberrant circulation of mobilized PB CD34(+) cells. Reexpression of a maximally activated alpha4 beta1 integrin on mobilized PB CFC removed from the mobilizing in vivo milieu may contribute to the early engraftment of mobilized PBPC.     

MaMi, a human endometrial stromal sarcoma cell line that constitutively produces interleukin-6, interleukin-8, and monocyte chemoattractant protein-1

BACKGROUND: Uterine endometrial stromal sarcoma is a rare neoplasm with a morphology that closely resembles that of the proliferative endometrial stroma. To understand its pathologic characteristics, we established a novel cell line, MaMi, from a primary culture of an endometrial stromal sarcoma obtained from a 65-year-old Japanese woman. METHODS: We observed the morphology of MaMi cells and performed immunohistochemical analysis on the primary tumor and transplants in nude mice. Prolactin, insulin-like growth factor-binding protein-1, interleukin (IL)-6, IL-8, monocyte chemoattractant protein-1, intercellular adhesion molecule-1, E-selectin, vascular cell adhesion molecule-1, and fibronectin production in the culture medium of MaMi cells were also examined. RESULTS: MaMi cells were shown to exhibit a fibroblast-like morphology in vitro, and they adopted a more elongated appearance in response to 12-O-tetradecanoyl phorbol-13-acetate (TPA). On injection into nude mice, the cells gave rise to subcutaneous tumors. Immunohistologically, both the primary tumor and MaMi cell-induced tumors stained positively with antibodies to neuron-specific enolase or vimentin. MaMi cells constitutively produced IL-6, IL-8, and monocyte chemoattractant protein-1 in vitro. Interleukin-1beta, (100 pmol/L), tumor necrosis factor-alpha (1 nmol/L), and lipopolysaccharide (1 microg/mL) each increased the release of IL-6, IL-8, and monocyte chemoattractant protein-1 by MaMi cells. TPA (10 nmol/L) also stimulated the production of IL-6 and IL-8 by these cells, but inhibited that of monocyte chemoattractant protein-1. CONCLUSIONS: We demonstrated that MaMi cells closely resemble proliferative endometrial stromal cells not only morphologically, but also functionally. This cell line may prove valuable in understanding the role of cytokines produced by tumor cells in the pathogenesis of endometrial stromal sarcoma and may also be useful as an in vitro model of functioning endometrial stromal cells.     

The ex vivo expansion capacity of normal human bone marrow cells is dependent on experimental conditions: role of the cell concentration, serum and CD34+ cell selection in stroma-free cultures

The present study was conducted to establish defined culture conditions for ex vivo expansion of normal human bone marrow cells. We investigated the role of three experimental expansion parameters: the cell concentration in the initial culture medium, the role of animal serum, human plasma and serum-free substitute, and the expansion potential of mononucleated cells (MNC) versus CD34+ cells. Cells were cultured in suspension with stem cell factor (SCF), IL3, IL6 and Erythropoietin (Epo) for 10 days. 1) Reducing the cell concentration from 3 x 10(4) to 1.5 x 10(3)/ml increased total cell expansion almost 20 fold, progenitor expansion more than 3 fold, and the maintenance of long term culture-initiating cells (LTC-IC). 2) In medium containing a serum-free substitute, total and CD34+ cell expansion was 3 times greater than in medium containing 1-10% human AB plasma or 25% animal serum. 3) The expansion potential of selected CD34+ cells was significantly greater than that of the total MNC population. However, taking into account the cell loss due to CD34+ selection, the overall results for quantitative expansion in relation to the initial number of MNCS favor the use of non-selected MNCS. 4) SCF + IL3 + IL6 was clearly the best combination of early cytokines for LTC-IC maintenance, with or without lineage-restricted cytokines, whereas the presence of IL1 beta in any combination augmented the decrease in LTC-IC. Addition of G-CSF to the medium resulted in 1 log increase in total cell expansion and a 2-fold increase in CFU-GM expansion. Addition of Epo always induced a dramatic proliferation of erythroid cells (up to 2000 fold) as well as of CFU-GM (up to 4 fold), without exhausting the LTC-IC pool. We concluded that the expansion of hemopoietic cells for clinical purposes needs establishment of controlled, reproducible and reliable culture conditions.     

Cell culture bags allow a large extent of ex vivo expansion of LTC-IC and functional mature cells which can subsequently be frozen: interest for a large-scale clinical applications

The aim of this study was to evaluate the ex vivo expansion of normal CD34+ cells in gas-permeable polypropylene bags suitable for clinical use. Cells were cultured for 14 days in serum-free medium supplemented with SCF, IL3, IL6, FLT3-1, G-CSF + MGDF or Epo. The bags supported the expansion of hematopoietic cells in a similar manner to small scale well or flask systems, allowing mean expansions of up to 2193-fold for total nucleated cells, 140-fold for CFU-GM and 66-fold for LTC-IC. Increasing the initial cell concentration from 5 x 10(3) to 1 x 10(5)CD34+ cells/ml induced the production of granulocytic cells with terminal differentiation while simultaneously decreasing the overall extent of expansion of the white blood cells produced. We tested the phagocytic activity and oxidative metabolism of the white blood cells produced. The percentage of phagocytic cells was 39+/-0.5% in expanded cultures derived from fractions initiated at 5 x 10(3), 10(4) or 10(5) cells/ml and 45+/-6% in cultured cells obtained from starting fractions containing 5 x 10(4) cells/ml, as compared to 58+/-4% in normal controls. A study of the potential for oxygen-dependent microbe killing showed that the expanded cells produced H2O2, although in lesser quantities than control cells. We subsequently investigated the possibility of freezing expanded cells. Total cell recovery after thawing was 45+/-4%, while recoveries of progenitors and stem cells ranged from 65 to 90%, without any influence of the initial cell concentration. This new approach could be of major interest for clinical practice, as it would allow evaluation of the quality of a graft prior to its infusion and employs experimental conditions which meet the criteria for potential clinical use.     

Fast modulation of glutamate and GABA receptor--mediate electrophysiological responses by glucocorticoid]

Endothelin (ET) is a powerful vasoconstrictor and bronchoconstrictor peptide that may be involved in the pathogenesis of bronchial asthma. We have investigated the effect of ET on the secretion of IL-6, IL-8, GM-CSF and G-CSF in a bronchial epithelial cell line (BEAS-2B). Incubation of BEAS-2B cells with ET-1 (10(-13) to 10(-7) M) for 4 h caused dose-related increases in the release of IL-8 (68% increase above control, P < 0.001) and IL-6 (43% increase above control, P < 0.001), compared to untreated control cells. After 48 h incubation, ET-1 also increased the release of IL-8 by 35% (P < 0.001) and GM-CSF by 38% (P < 0.01). ET-1 had no significant effect on G-CSF release. ET-1 did not induce cell proliferation at 24 or 48 h. Since ET-immunoreactive materials are expressed in epithelial cells in asthma, it is possible that ET-1 of epithelial origin may act in a paracrine or autocrine fashion on airway epithelial ET receptors to stimulate IL-8, IL-N6 and GM-CSF release. Thus, ET-1 may play a role in the regulation of the cytokine responses involved in inflammation of the airway mucosa.     

A double-blind comparison of the efficacy and safely of paroxetine and imipramine in the treatment of depression with dementia

Marrow stromal cells of patients treated by autologous bone marrow transplantation (ABMT) for malignancies have been assessed for their ability to secrete granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), stem cell factor (SCF), leukemia inhibitory factor (LIF), interleukin-6 (IL-6), transforming growth factor beta1 (TGFbeta1) and macrophage inflammatory protein-1alpha. (MIP-1alpha). Long-term marrow cultures were established from 10 patients prior to and 3 months after ABMT, from 7 patients 1 yr after ABMT and from 11 controls. Cytokines in culture supernatants of stromal layers (SL) were evaluated by enzyme-linked immunosorbent assay (ELISA). Significant differences between patient groups and controls were apparent in baseline production of GM-CSF, SCF, MIP-1alpha and TGFbeta1. After IL-1beta addition in cultures, G-CSF production was reduced in pretransplant and post-transplant patients compared to controls. The production of TGFbeta1, LIF, IL-6 and more particularly SCF were reduced in post-transplant patients, while elevated levels of GM-CSF and MIP-1alpha were observed in these patients only when the values were corrected for the number of cells growing in the SL. These results indicate a prolonged stromal defect in growth factor production following ABMT for the early-stage acting cytokines IL-6, LIF and SCF as well as for G-CSF, but not for GM-CSF, while the production of the 2 inhibitors shows different pathways.     

Human CD34(+) bone marrow cells regulate stromal production of interleukin-6 and granulocyte colony-stimulating factor and increase the colony-stimulating activity of stroma

Cytokines produced by stromal cells induce the proliferation and differentiation of hematopoietic cells in the marrow microenvironment. We hypothesized that cross-talk between hematopoietic cells at different stages of differentiation and stromal cells influences stromal cytokine production and is responsible for maintaining steady-state hematopoiesis and responding to stress situations. We show that coculture of primitive CD34(+) cells in contact with or separated by a transwell membrane from irradiated human bone marrow stromal layers induces a fourfold to fivefold increase in interleukin-6 (IL-6) and granulocyte colony-stimulating factor (G-CSF) levels in the stromal supernatant (SN) during the first week. Levels of both cytokines decreased to baseline after coculture of CD34(+) cells for 3 to 5 weeks. Coculture of more mature CD15(+)/CD14(-) myeloid precursors induced only a transient 1.5- to 2-fold increase in IL-6 and G-CSF at 48 hours. Neither CD34(+) nor CD15(+)/CD14(-) cells produced IL-6, G-CSF, IL-1beta, or tumor necrosis factor alpha. When CD34(+) cells were cultured in methylcellulose medium supplemented with cytokines at concentrations found in stromal SN or supplemented with stromal SN, a fourfold to fivefold increase in colony formation was seen over cultures supplemented with erythropoietin (EPO) only. When cultures were supplemented with the increased concentrations of IL-6 and G-CSF detected in cocultures of stroma and CD34(+) cells or when CD34(+) cells were cocultured in methylcellulose medium in a transwell above a stromal layer, a further increase in the number and size of colonies was seen. The colony-forming unit-granulocyte-macrophage-stimulating activity of stromal SN was neutralized by antibodies against G-CSF or IL-6. These studies indicate that primitive CD34(+) progenitors provide a soluble positive feedback signal to induce cytokine production by stromal cells and that the observed increase in cytokine levels is biologically relevant.     

A combination of stem cell factor and granulocyte colony-stimulating factor enhances the growth of human progenitor B cells supported by murine stromal cell line MS-5

We have developed a long-term culture system using the murine bone marrow stromal cells MS-5 to support the growth of progenitor B cells with CD34-, CD10+, CD19+, and cytoplasmic mu chain (C mu)-negative surface phenotype from human CD34+ cells purified from umbilical cord blood (CB). When 10(3) CD34+ cells/well were seeded on MS-5 stromal cells at the beginning of culture in the absence of exogenously added cytokines, progenitor B cells first appeared after 14 days, and the maximal cell production was achieved during the 6th week of culture. Intriguingly, the addition of recombinant human stem cell factor (rhSCF) and granulocyte colony-stimulating factor (rhG-CSF), but not rhIL-7, strikingly enhanced the growth of progenitor B cells from CB CD34+ population cultured on MS-5 stromal cells. The culture of progenitor B cells could be maintained until the 6th week of culture when some cells were revealed to have a C mu phenotype, and a small number of cells had immunoglobulin mu chain on their cell surface in the presence of both rhSCF and rhG-CSF. When CD34+ cells were cultured physically separated from the stromal layer by membrane, supportive effects of MS-5 stromal cells for the growth of progenitor B cells were not observed. These results suggest that the present culture system could generate progenitor B cells to proliferate from CB CD34+ cells, that some of these progenitor B cells could differentiate into immature B cells in conjunction with rhSCF and rhG-CSF, and that a species-cross-reactive membrane-bound factor(s), which stimulates early human B lymphopoiesis, may exist in MS-5 stromal cells. Further studies are required to investigate the mechanism how rhG-CSF acts on progenitor B cells to allow their proliferation and differentiation.     

Interferon-gamma and interleukin-4 reciprocally regulate the production of monocytes/macrophages and neutrophils through a direct effect on committed monopotential bone marrow progenitor cells

We studied the direct effects of interferon-gamma (IFN-gamma) in single cell colony assays of CD34+HLA-DR++ bone marrow progenitor cells stimulated by either granulocyte-colony-stimulating factor (G-CSF), interleukin(IL)-3, granulocyte/macrophage-colony-stimulating factor (GM-CSF), combinations of these CSF or medium conditioned by the 5637 human bladder carcinoma cell line. In this culture system IFN-gamma stimulated monocytic colonies (CFU-M) no matter which CSF or CSF combination was used to support them and inhibited granulocytic colonies (CFU-G) if they were generated in the presence of G-CSF. IL-4 antagonized the myelopoietic effects of IFN-gamma: the IFN-gamma-induced suppression of G-CSF-supported CFU-G, as well as the stimulation of CFU-M, were reversed by IL-4. In all cultures, IFN-gamma had a limited, but statistically non-significant, inhibitory effect on CFU-GM, which was not affected by the presence of IL-4. These data show that IFN-gamma and IL-4 reciprocally regulate the generation of myeloid cells involved in humoral (neutrophils) and cellular (macrophages) immune responses through a direct effect on monopotential myeloid progenitor cells.     

Conditional immortalization of human endometrial stromal cells with a temperature-sensitive simian virus 40

The study of immortalization and other alterations associated with neoplastic transformation of endometrial stromal cells is important to understanding the development of uterine sarcomas and mixed tumors. Because stromal cells are important regulators of associated epithelial cells, alterations in the regulation of stromal cell proliferation that influence epithelial cells may also contribute to the development of endometrial carcinomas. To study immortalization and associated phenotypic and genetic alterations of human endometrial stromal cells, cultures were transfected with a plasmid containing an ori-, temperature-sensitive mutant SV40, A209 (tsSV40). Morphologically transformed colonies were selected and propagated at the permissive temperature until they entered 'crisis'. In contrast to human fibroblasts, every clone tested was immortalization competent. The frequency of immortalization was approximately 1 x 10(-6). One uncloned and six cloned cell lines escaped from crisis and appear to be immortal. Two clones, M4 and B10T1, were selected for further study. Immortalization is conditional; proliferative arrest occurs at the restrictive temperature for large T antigen function. Furthermore, withdrawal of the large T antigen results in expression of the senescent phenotype of enlarged, flattened cells. Colony-forming efficiency at the restrictive temperature was undetectable. Immortalization is also associated with several genetic alterations. The DNA content of tsSV40 transfected cells was either diploid or tetraploid in the precrisis stage of proliferation, but became aneuploid upon immortalization. Several structural rearrangements of chromosomes were detected in the immortalized stromal cells which differ from those found in SV40 immortalized fibroblasts. Although their capacity for anchorage-independent proliferation (AIP) is variable, tsSV40-immortalized endometrial stromal cells have a higher capacity for AIP than their tsSV40-transfected progenitor cells in the period of proliferation prior to 'crisis'.     

Frequency analysis of human primitive haematopoietic stem cell subsets using a cobblestone area forming cell assay

Stroma-dependent long-term bone marrow cultures (LTBMC) assay the ability of primitive haematopoietic stem cells (HSC) for long-term production of clonable progenitors. We have developed a limiting dilution type LTBMC assay allowing frequency analysis of transiently repopulating HSC and long-term culture initiating cells (LTC-IC) without the necessity to replate large numbers of wells. Normal or 5-FU-treated Ficoll bone marrow cells (BMC), or BMCs sorted on CD34 or HLA-DR expression, or Rh123 retention, (input range 40-70,000 CFU-GM/BFU-E/10(5) cells) were plated at limiting dilution on unirradiated adherent layers formed by a novel murine preadipose cell line (FBMD-1). The percentage of wells with at least one phase-dark haematopoietic clone (cobblestone area, CA) beneath the stromal layer was weekly determined for at least 8 weeks, and CA-forming cell (CAFC) frequencies were calculated using Poisson statistics. Parallel LTBMCs of the same samples were weekly assessed for supernate CFU-GM/BFU-E production. Weekly addition of rhIL-3 with rhG-CSF supported a high average clonogenic output per CA and dramatically increased CA size, but did not significantly alter the apparent CAFC frequency. The generation of CFU-GM per CA was constant over a period of 6 weeks with weekly means of eight normal BM samples, ranging between 5-16. At week 6 the mean CAFC frequency was 29 (1 SEM, 8.8)/10(5). Early appearing CAFC were highly sensitive to 5-FU, and were contained over the full Rh123 and HLA-DR fluorescence profile of CD34pos cells, whereas CAFC week 5-8 were predominantly contained in the CD34pos Rh123dull HLA-DRlow fraction in agreement with previously reported LTC-IC characteristics. In conclusion, the CAFC assay enumerates LTC-IC using a direct visual endpoint and allows study of LTC-IC heterogeneity with respect to progenitor cell generation per stem cell clone in various haematologic diseases.     

Lymphocyte activation and cytokine production in autoimmune hemolytic anaemia (AIHA)

We studied 16 patients affected by autoimmune hemolytic anaemia (AIHA), both idiopathic and associated with other diseases (B and T lymphoma, B hepatitis, gastric carcinoma, systemic lupus erythematosus) or alpha-methyldopa therapy, in order to value T- and B-cell activation. We determined the count of T- and B-cell subsets in peripheral blood, the proliferative response of peripheral blood lymphocytes (PBL) to phytohemagglutinin (PHA) and to pokeweed mitogen (PWM), the percentage of CD25+ cells in culture and interleukin (IL)-1alpha, IL-2, IL-4, tumor necrosis factor (TNF)alpha and soluble IL-2 receptor (sIL-2R) levels in sera and in culture. Except for an increase in CD4+ and CD8+ T cell number in a case of AIHA associated with a T lymphoma and an increase in the percentage of CD5+ and PCA1+ B cells in two cases of AIHA associated with B lymphoma and with SLE, no further data showed a relationship with the disease possibly associated with AIHA, so both idiopathic and secondary AIHA cases were analyzed together. CD4+ T cells were reduced in number in 9 cases, while CD8+ T cells were reduced in 6 cases. The percentage of CD5+ B cells was increased in 5 cases. The percentage of PCA1+ cells was increased in all cases (mean +/- sd: 18 +/- 22 vs 0,2 +/- 1 in controls). The average PBL proliferative response to PHA was reduced (S.I. 71 +/- 55 vs 138 +/- 45 in controls) as well as that to PWM (S.I. 27 +/- 21 vs 75 +/- 24 in controls), despite IL-2 high levels, in all cases, in both sera (mean +/- sd: 648 +/- 351 pg/ml vs 16 +/- 4 pg/ml in controls) and culture supernatants (mean +/- sd: 1045 +/- 677 pg/ml vs 195 +/- 51 pg/ml in controls). In PHA stimulated cultures the percentage of CD25+ cells was reduced (mean +/- sd: 37 +/- 18 vs 63 +/- 14 in controls), sIL-2R levels were like controls in 7 cases. In sera sIL-2R levels were increased in all cases (mean +/- sd: 1256 +/- 465 U/ml vs 256 +/- 114 U/ml in controls), IL-1alpha was increased in all cases too, while IL-4 levels were increased only in 7 cases. Linear regression analysis generally showed a low relationship between S.I. and IL-2, IL-4 and sIL-2R levels in supernatants of PHA stimulated culture as well as between S.I. and the percentage of CD25+ cells. Taken together these data suggest a state of B- and T-cell hyperactivation in AIHA. The low PBL proliferative response in vitro, explained in previous studies as a temporary functional exhaustion, might be itself a sign of the complete lymphocyte activation occurring in vivo in AIHA.