Biomedical subjects

U Kalina

Publications and source records attributed to U Kalina.

At least 19 recordsLinked to original sources

PubMed2002

Prednisolone induces interleukin-18 expression in mononuclear blood and myeloid progenitor cells.

OBJECTIVE: Interleukin (IL)-18 is involved in host defense mechanisms and inflammatory diseases, among them rheumatoid arthritis (RA). High levels of IL-18 expression in RA joints are contrasted by reduced IL-18 expression in RA peripheral blood mononuclear cells (PBMC). Here, we investigated a putative IL-18 regulating role of corticosteroids. METHODS: IL-18 transcript and protein levels in PBMC from untreated and prednisolone treated RA patients, and from healthy donors were assessed by semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) and immunoblotting. IL-18 regulation was determined in PBMC and U937 cells upon exposure to prednisolone in vitro by RT-PCR and Northern Blot analysis, by ELISA in cell culture supernatants, and in transiently transfected THP-1 cells by IL-18 promoter activity luciferase assays. RESULTS: In RA PBMC, IL-18 transcript levels were dose dependently restored, in parallel with administered prednisolone treatment, to subnormal levels. The corresponding intracellular IL-18 deposits in contrast were depleted. In cultured PBMC and promonocytic cell lines, prednisolone up-regulated IL-18 transcription in parallel with increasing the IL- 18 protein release into cell culture supernatants. CONCLUSION: Prednisolone increases IL-18 expression and release in PBMC and monocytic cell lines.

Adult↗

PubMed2001

Murine M2-10B4 and SL/SL cell lines differentially affect the balance between CD34+ cell expansion and maturation.

The ability of bone marrow stroma to modulate hematopoietic progenitor cell expansion is of considerable interest for gene transfer strategies and transplantation of limited stem cell numbers. We compared the capacity of 2 murine stromal cell lines to affect the balance between maturation and proliferation of human CD34+ cells in short-term expansion cultures. In 7-day serum-free cultures, cytokine-induced amplification of granulocyte-macrophage colony-forming cells (CFC-GM), erythroid burst-forming units (BFU-E), and total cells was significantly increased by the presence of genetically engineered Sl/Sl and M2-10B4 stromal cells in a 1:1 ratio (Sl/M2 cells) compared with stroma-free cultures (P < .05). Sl/M2 cultures generated 21-fold more mature CD15+ cells than stroma-free cultures, without further amplifying the number of CD34+ cells. The addition of serum led to a further increase of CFC-GM, total cells, and CD15+ cells, whereas BFU-E were no longer maintained. Pure Sl/Sl stromal layers were likewise superior to stroma-free cultures in expansion of CD34+ cells and total cells when serum was present. However, the differentiation of CD34+ cells was less pronounced in Sl/Sl cultures compared with Sl/M2 layers, as demonstrated by a lower content of CD15+ cells. Neutralization experiments revealed differential contributions of Flt3 ligand and thrombopoietin to the support of total cell and CFC expansion by Sl/M2 and Sl/Sl stromal feeders.

Adjuvants, Immunologic↗

PubMed2001

Transforming growth factor-beta1 interferes with thrombopoietin-induced signal transduction in megakaryoblastic and erythroleukemic cells.

OBJECTIVE: Thrombopoietin (TPO) and transforming growth factor-beta(1) (TGF-beta(1)) have been shown to exert opposite effects on proliferation and megakaryocytic differentiation of hematopoietic cells. To determine whether TGF-beta(1) interferes directly with TPO-induced signal transduction in hematopoietic cells, we compared the regulatory effects in the TPO-responsive cell lines Mo-7e and HEL. MATERIALS AND METHODS: The cells were stimulated by 100 ng/mL TPO and/or 100 ng/mL TGF-beta1 and analyzed for proliferation (3H thymidine incorporation), viability (trypan blue exclusion), and protein expression and phosphorylation (Western blot). RESULTS: TPO enhanced the proliferation of Mo-7e cells as determined by 3H-thymidine incorporation, whereas TGF-beta1 suppressed baseline cell growth and antagonized the proliferative effect of TPO. TPO-induced proliferation also was reduced by a specific inhibitor of the mitogen-activated protein kinase (MAPK) pathway (PD098059), which inhibits activation of the MAPK extracellular signal-regulated kinases (ERK) ERK1 and ERK2, and AG490, an inhibitor of Janus kinase-2, which completely blocked TPO-induced proliferation. As demonstrated by Western blotting, TGF-beta1 reduced the TPO-stimulated ERK1/ERK2 and STAT5 phosphorylation in Mo-7e and HEL cells. This effect was completely reversed by preincubation with a tyrosine phosphatase inhibitor (Na3VO4), which suggests that TGF-beta1 activated a phosphatase. Although STAT3 also was activated by TPO, STAT3 activation remained unaltered by TGF-beta1. CONCLUSION: Taken together, these data suggest that TGF-beta1 modulates TPO-mediated effects on megakaryocytic proliferation by interfering with TPO-induced signal transduction, particularly by reducing the activities of MAPK ERK1/ERK2 and STAT5.

Cell Differentiation↗

PubMed2001

TGF beta-induced SMAD2 phosphorylation predicts inhibition of thymidine incorporation in CD34+ cells from healthy donors, but not from patients with AML after MDS.

Cells from patients with MDS-derived AML display heterogeneous proliferative responses to transforming growth factor beta (TGF beta). We analyzed growth inhibition and SMAD2 phosphorylation by TGF beta in CD34+ cells from nine patients, as compared to normal controls. While TGF beta consistently inhibited thymidine incorporation of normal cells (41% of control, P < 0.05), cells from patients with AML were growth-inhibited in only four of seven cases (40%), whereas TGF beta stimulated thymidine incorporation in the three other samples (166%). Remarkably, TPO reverted the stimulatory effect of TGF beta to profound growth inhibition. Upon exposure to TGF beta, SMAD2 protein was phosphorylated in normal CD34+ cells (n = 3), CD34+ leukemic blasts from all examined patients with AML (n = 4), and in the myeloid leukemic cell lines M-07e and HEL. TGF beta inhibited TPO-mediated thymidine incorporation, cell proliferation and survival in all samples analyzed. In M-07e cells and CD34+ cells from healthy donors, this inhibition was enhanced by an antagonist of JAK2 (AG490), but not a MEK-1 antagonist (PD098059). Conversely, in CD34+ cells from a patient with AML, both AG490 and PD098059 significantly enhanced TGF beta-mediated suppression of TPO-induced thymidine incorporation. Thus, in MDS-derived AML, altered responses to TGF beta may be due to defects downstream of SMAD2 and may involve MAPK activation.

Acute Disease↗

PubMed2001

Expression of interleukin-18 and its monokine-directed function in rheumatoid arthritis.

OBJECTIVES: To investigate the expression of and monokine induction by interleukin 18 (IL-18; also called interferon-gamma inducing factor, IGIF), in peripheral blood mononuclear cells (PBMC) and cultured synoviocytes from rheumatoid arthritis (RA) patients. METHODS: We carried out IL-18 Western blotting and semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) of cytokines in PBMC [IL-18, IL-1beta and tumour necrosis factor alpha (TNF-alpha)] and long-term cultured fibroblast-like synoviocytes (FLS) [IL-18, IL-1beta, TNF-alpha, IL-6, interferon gamma (INF-gamma) and [granulocyte-macrophage colony stimulating factor (GM-CSF)] from RA patients and controls. FLS were isolated from RA synovial membranes (FLS(SM)) and RA synovial fluids (FLS(SF)), osteoarthritis (OA) FLS(SM) and FLS(SF) from spondyloarthropathy patients. FLS were characterized by fluorescence-activated cell sorting of the FLS. PBMC and FLS from RA patients and control subjects were stimulated with recombinant human IL-18 and IL-1beta (rHuIL-18/rHuIL-1beta), and TNF-alpha, IL-1beta and MMP-1 were measured by ELISA in supernatants. RESULTS: Constitutive expression of IL-18 mRNA was significantly reduced whereas that of TNF-alpha was enhanced in RA PBMC. Persistent low expression of IL-18, TNF-alpha, GM-CSF and IL-1beta was observed in RA and OA FLS(SM) as well as spondyloarthropathy FLS(SF). In contrast, high constitutive expression of IL-18 in FLS (CD90/Thy-1- and CD54-positive, CD14- and CD86-negative), accompanied by persistent high levels of TNF-alpha, GM-CSF and IL-1beta expression, was restricted to synovial fluid-derived FLS obtained from RA patients. IFN-gamma was not detectable in any culture, but IL-6 mRNA was equally expressed in all FLS cultures. rHuIL-18 was effective in stimulating TNF-alpha and IL-1beta secretion in PBMC from healthy controls, but failed to stimulate TNF-alpha and IL-1beta secretion from PBMC in 11 of 12 RA patients, and all FLS cultures. rHu-IL-1beta, but not rHu-IL-18, induced interstitial collagenase (MMP-1) in FLS. CONCLUSIONS: Persistent high production of proinflammatory cytokines in RA-FLS(SF) may be relevant for chronic progression in RA synovitis. Levels of TNF-alpha and IL-1beta expression are increased in RA-FLS(SF), but are independent of IL-18. The pathological function of enhanced IL-18 expression in RA-FLS(SF) remains to be further elucidated.

Adult↗

PubMed2000

IL-18 activates STAT3 in the natural killer cell line 92, augments cytotoxic activity, and mediates IFN-gamma production by the stress kinase p38 and by the extracellular regulated kinases p44erk-1 and p42erk-21.

IL-18 is a regulator of NK cell function which utilizes the serine-threonine IL-1R-associated kinase signal transduction pathway and may activate additional not yet characterized signaling pathways. Here we evaluated IL-18-mediated signal transduction using the human NK cell line NK92 as a model. NK92 cells were shown by RT-PCR to express all three IL-18 receptor chains (IL-18R, accessory protein-like chain, IL-18-binding protein). Stimulation by IL-18 strongly enhanced tyrosine phosphorylation of STAT3 and of the mitogen-activated protein kinases (MAPK) p44erk-1and p42erk-2. In contrast, STAT5 was not activated. The cytolytic activity of NK92 against K562 target cells, which was augmented in a dose-dependent manner by IL-18 in the presence of trace amounts of IL-2, was suppressed by the specific inhibitors of MAPK pathways (PD098059 and SB203580). Similarly, the stimulatory effect of IL-18 on IFN-gamma protein production, given in conjunction with IL-2, was counteracted by inhibition of MAPK. IL-18 alone failed to stimulate IFN-gamma protein production despite inducing expression of IFN-gamma mRNA. IL-2 alone stimulated neither IFN-gamma mRNA expression nor IFN-gamma protein production. IL-18 did not stimulate proliferation of NK92 cells, either alone or in combination with IL-2 or IL-12. Inhibition of the MAPK pathway did not significantly alter the IL-2- and IL-12-induced proliferation of NK92 cells, whereas the Janus kinase/STAT pathway inhibitor AG490 strongly suppressed proliferation. MAPK activation appears to play a prominent role in IL-18 signaling, being involved in transcription and translation of IL-18-induced IFN-gamma mRNA and IL-18-induced cytolytic effects. In contrast, proliferation of NK92 cells is not affected by MAPK p44erk-1 and p42erk-2.

Adjuvants, Immunologic↗

PubMed2000

Effect of treatment with amifostine used as a single agent in patients with refractory anemia on clinical outcome and serum tumor necrosis factor alpha levels.

Amifostine increases in vitro burst-forming unit-erythroid and colony-forming unit-granulocyte/granulcoyte-macrophage cultured from bone-marrow cells from patients with myelodysplastic syndrome (MDS). Several small clinical studies give divergent informations about the potential of amifostine as single agent to improve hematopoiesis in MDS patients. In these studies, patients with refractory anemia (RA), RA with excess of blasts (RAEB), and RAEB in transformation (RAEB-T) were analyzed together, resulting in response rates varying from 8% to 30%. The present multi-center study evaluated whether treatment with amifostine is of clinical benefit in patients with RA who are transfusion dependent. The effect on transfusion frequency as well as on platelets and absolute neutrophil count (ANC) was examined in 14 patients with RA [median age 67 years (55-72 years), male:female 9:5]. Four treatment cycles were planned, each consisting of intravenous amifostine at 200 mg/m2/day three times per week followed by a 2-week interval. Since tumor necrosis factor (TNF) alpha is a main suppressive cytokine for hematopoiesis in RA patients, serum samples for analyzing endogenous levels of TNF alpha were collected prior to the study and after four treatment cycles. In three patients (21%), reduced transfusion requirement with prolongation of the transfusion interval from 4 weeks to 8 weeks (two patients) and 4 weeks to 6 weeks was seen. An increase in ANC from 400/microliter to 2600/microliter and 200/microliter to 3400/microliter was observed in two patients. Platelets increased from 129,000/microliter to 277,000/microliter in an additional patient. In one patient, disease progression from RA to RAEB was observed. Serum TNF alpha levels were increased in MDS patients compared with normal controls (18.8 pg/ml vs 9.1 pg/ml), and there was no change during the treatment with amifostine (17.5 pg/ml). In conclusion, treatment with amifostine as a single agent was of limited benefit in patients with RA. The serum TNF alpha levels were unchanged during treatment with amifostine in RA patients.

Aged↗

PubMed2000

Alteration of c-mpl-mediated signal transduction in CD34(+) cells from patients with myelodysplastic syndromes.

OBJECTIVE: Megakaryocytic differentiation is frequently defective in patients with myelodysplastic syndromes (MDS). As underlying mechanisms, deregulated thrombopoietin receptor (c-mpl)-mediated signaling pathways have been suggested. This study therefore examined whether the impaired signaling in MDS and AML cells includes alterations of c-mpl itself or postsignaling events. METHODS: Bone marrow-derived CD34(+) cells from healthy donors, patients with MDS (RA, RAEB-T), and patients with AML after MDS were isolated by MACS. Expression of c-mpl cDNA was studied by RT-PCR. Thrombopoietin dependent activation of STAT proteins and MAP Kinase p42(erk-2)/44(erk-1) was analyzed by Western blot. RESULTS: Both splicing isoforms of c-mpl (c-mpl-p and c-mpl-k) were expressed in all of the CD34(+) cells examined. Analysis of the c-mpl cDNA revealed no sequence abnormality. We show c-mpl dependent activation of the transcription factors STAT3 and STAT5 as well as MAP Kinase p42(erk-2)/44(erk-1) in CD34(+) cells from healthy individuals. Cells derived from RA patients revealed low basal levels of phosphorylated STAT3 and STAT5 molecules. This phosphorylation was enhanced by stimulation with recombinant thrombopoietin (PEG-rHuMGDF). STAT1 failed to be activated by PEG-rHuMGDF in CD34(+) cells from healthy donors as well as from patients with MDS. In RAEB-T and AML M7 the constitutive expression levels of STAT1, 3, 5, and MAPK were markedly upregulated, resulting in a strong activation of STAT3 and 5 by PEG-rHuMGDF. Despite its high expression, the level of MAPK phosphorylation was not increased in RA or RAEB-T compared to the normal control, and was completely undetectable in AML M7. CONCLUSION: These results suggest that the defective megakaryopoiesis in MDS is not caused by a lack of c-mpl and that STAT3 and STAT5 may contribute to the malignant phenotype of the leukemic cells.

Anemia, Refractory↗

PubMed2000

Genomic organization and regulation of the human interleukin-18 gene.

The human interleukin(IL)-18 is a key regulator of interferon(IFN)-gamma production and T-cell differentiation. Here we report the complete genomic structure and characterization of the 5'untranslated promoter region of the human IL-18 gene. The gene is composed of six exons and five introns, spanning approximately 19. 5kb. Promoter activity of the 5'-flanking region was investigated with a luciferase reporter gene assay. Transient transfection studies demonstrate a constitutive expression of the IL-18 gene in monocytic U937 and THP-1 cells. For this constitutive expression at least 92 base pairs of the promoter region are essential as shown by consecutive 5' promoter deletions in both cell types. DNA protein binding experiments revealed specific binding of activated signal transducer and activator of transcription factor-5 (STAT5) but not of STAT3 to three consensus sequences upstream in the promoter region. Cotransfection of STAT5 resulted in increased induction of the IL-18 promoter in the U937 and THP-1 cells.

Base Sequence↗

PubMed2000

Defective megakaryocytic development in myelodysplastic syndromes.

Megakaryocytic proliferation and differentiation is typically abnormal in patients with myelodysplastic syndromes (MDS). The underlying mechanisms for this finding are not known, but may involve defects at the level of the thrombopoietin-receptor (c-mpl) or post-receptor signaling pathways in megakaryocyte progenitor cells. Premature apoptosis of the bone marrow cells and inhibitory effects of cytokines such as tumor necrosis factor alpha have been implicated as contributing to altered megakaryopoiesis in MDS, but their significance remains unclear. The availability of thrombopoietin (TPO) has facilitated more detailed analysis of megakaryocytic biology using several experimental in-vitro systems. However numerous studies have shown that the developmental abnormalities of MDS megakaryocytes could not be corrected by TPO. Increasing investigations are being extended to the evaluation of signal transduction pathways of c-mpl both in cell lines and human hematopoietic cells in order to identify the molecular mechanisms responsible for the defective megakaryocytic development in MDS.

Cell Differentiation↗

PubMed2000

Interleukin-18 stimulates HIV-1 replication in a T-cell line.

Interleukin-18 (IL-18) is a recently identified proinflammatory cytokine. Its ability to induce interferon-g suggests a potential virustatic effect. On the other hand, it stimulates NFkB - an activator of HIV replication. Recently, stimulation of HIV-1 in monocytic cells has been demonstrated. In the present study, the influence of IL-18 on HIV-1 replication in lymphatic cells was investigated. Hut78 cells were infected with HIV-1 in the presence of recombinant human IL-18 expressed either in E. coli or eucaryotically by baculovirus in Sf9 cells. HIV-1 replication was monitored by p24 ELISA and endpoint titration of culture supernatants on C8166 cells. The addition of IL-18 led to a 3- to 15-fold enhancement of HIV replication in Hut78 cells. By addition of neutralising monoclonal anti-IL-18 antibodies, this effect of IL-18 was reduced by 75%. Exposure of Hut78 to IL-18 prior to HIV infection could exclude the possibility that IL-18 promotes infection of cells. Taken together, these data provide direct evidence for an IL-18-mediated enhancement of HIV-1 replication in lymphatic cells.

Animals↗

PubMed2000

Regulation of interleukin-18 (IL-18) expression in keratinocytes (HaCaT): implications for early wound healing.

Keratinocytes display a high basal level expression of IL-18. Tumor necrosis factor-alpha (TNF-alpha) mediated a large decrease in IL-18 mRNA levels in the human keratinocyte cell line HaCaT, which was accompanied by a subsequent accumulation of IL-18 protein in the cell culture supernatants, which was shown to be biologically active. By contrast, epidermal growth factor (EGF) and transforming growth factor-alpha (TGF-alpha), respectively, strongly decreased IL-18 mRNA expression in HaCaT keratinocytes in the absence of IL-18 protein release from the cells. Notably, a pre-treatment of the cells with EGF, or TGF-alpha clearly attenuated TNF-alpha-induced IL-18 protein, release and bioactivity. For the in vivo situation of cutaneous wound repair, we observed an increase in IL-18 protein, 10 hours post-wounding, that closely correlated to infiltration of neutrophils which are known as producers of TNF-alpha. Our data suggest that bioactive IL-18 might be tightly counter-regulated by platelet- and neutrophil-derived factors at the onset of repair.

Blood Platelets↗

PubMed1999

Quantification of human interleukin 18 mRNA expression by competitive reverse transcriptase polymerase chain reaction.

Interleukin 18 (IL-18) is a recently identified cytokine, originally called interferon gamma inducing factor, due to its capacity to induce interferon gamma production in Th1 type cells. IL-18 is expressed by a wide variety of cell types including mononuclear phagocytes, osteoblasts, keratinocytes and adrenal cortex cells. To quantify human IL-18 mRNA expression in small-scale cell samples the authors developed a competitive reverse transcriptase polymerase chain reaction using a competitive template as an internal standard. This assay was demonstrated as a valid, sensitive and precise tool to quantify human IL-18 mRNA expression. IL-18 mRNA expression of primary peripheral blood monocytes, CD4(+)T cells, CD8(+)T cells, B cells and NK cells was assessed by competitive RT-PCR. Basal IL-18 expression could be detected in all types of peripheral blood mononuclear cells (PBMC). The kinetics of IL-18 mRNA expression in PBMC from healthy donors was defined in vitro after monocyte-specific (lipopolysaccharide LPS), T-cell-specific (anti-CD3) and polyclonal-unspecific stimulation (phytohaemagglutinin PHA). Only LPS led to a strong increase of IL-18 mRNA expression peaking after 2 h. These results indicate that IL-18 is expressed constitutionally by all major PBMC subtypes. However, only monocyte specific stimulation resulted in a significant induction of IL-18 mRNA expression suggesting activated monocytes e.g. in inflammation as the main source of IL-18 expression.

Binding, Competitive↗

PubMed1999

Characterization of defective megakaryocytic development in patients with myelodysplastic syndromes.

Megakaryocytic differentiation of progenitor cells was investigated in nine patients with low-risk myelodysplastic syndromes (MDS) (eight refractor anemia [RA] and one RA with ringed sideroblasts [RARS] and five patients with high-risk MDS (two RA with excess of blasts [RAEB] and three RAEB in transformation [RAEB-T]). Bone marrow-derived CD34+ cells were enriched to a purity of 87% +/- 2% (mean +/- SEM) and assayed in short-term suspension cultures in the presence of 10 ng/mL of PEGylated recombinant human megakaryocyte (MK) growth and development factor (PEG-rHuMGDF) and in addition to 50 ng/mL stem cell factor and 10 ng/mL interleukin-3. Cells of the megakaryocytic lineage were identified by flow cytometric analysis of CD42b (GP1b) and mature MKs by morphologic criteria. Transcription of c-mpl receptor-specific mRNA in the CD34+ cells of these patients was investigated by full-length reverse transcriptase polymerase chain reaction of the p form of c-mpl as well as of the alternative splice product c-mpl k. CD34+ cells from seven healthy bone marrow donors served as controls. Differentiation along the MK pathway was stimulated in five patients with RA. C-mpl mRNA was expressed in the CD34+ cells in all cases. In three low-risk patients the capacity for in vitro MK growth was absent or minimal even though mRNA for c-mpl receptor was detected in the CD34+ cells of this group as well. In patients with high-risk MDS, PEG-rHuMGDF stimulated in vitro MK growth from CD34+ cells in only one of five cases. As in the patients with low-risk MDS, c-mpl mRNA for both c-mpl p and c-mpl k splicing products was detected. These results indicate that the in vitro response to stimulation with c-mpl ligand discriminates between two groups of patients with low-risk MDS and that the observed defect in megakaryocytic development is unrelated to the level of c-mpl expression in both low-risk and high-risk MDS.

Antigens, Differentiation↗

PubMed1999

Counterregulation of interleukin-18 mRNA and protein expression during cutaneous wound repair in mice.

Recent work has suggested interleukin-18 to represent a proinflammatory cytokine that contributes to systemic and local inflammation. As the process of cutaneous wound healing crucially involves an inflammatory phase of repair, we investigated the regulation of interleukin-18 during the repair process. In non-wounded skin we observed high levels of interleukin-18 mRNA, whereas corresponding interleukin-18 protein was expressed only at low basal levels. Upon injury, we found a rapid and large induction of interleukin-18 protein expression, which is directly correlated with decreasing mRNA levels within the wound. Immunohistochemical analysis revealed different sites of expression in the wounded area, with keratinocytes as one major source of interleukin-18 production. The counterregulation of interleukin-18 mRNA and protein expression during wound repair in vivo might represent a general mechanism for interleukin-18 expressional regulation, as cytokine-stimulated keratinocytes exhibit a similar downregulation of interleukin-18 mRNA that is directly associated with increasing interleukin-18 protein levels in vitro. The rapid induction of interleukin-18 during wound healing suggests a role for interleukin-18 within the early phase of repair rather than a role in costimulation of interferon-gamma release from T cells, which are present in high numbers within the wounded area only during the late inflammatory phase of repair.

Animals↗

PubMed1999

Megakaryocytic growth in patients with refractory anemia is suppressed by treatment with interferon alpha.

IFN alpha alone or in combination with retinoids or haematopoietic growth factors has been used to treat patients with early MDS because of its properties as a differentiation inducing agent. We investigated whether treatment of patients with refractory anemia (RA) with IFN alpha (1.5x10(6) IU twice a week) and intermittent all-trans retinoic acid (ATRA, 25 mg/m2/d) influences in-vitro megakaryocytic (MK) proliferation and differentiation stimulated by PEG-rHuMGDF. Low-density non-adherent bone marrow (BM) cells from 8 patients with RA were assayed prior to any treatment other than supportive and after a period of 6 months of treatment. MK development was assayed in suspension cultures in the presence of PEG-rHuMGDF and SCF for 7 d using morphological criteria and flow cytometric analysis of CD42b (GP1b) positive cells. BM-cells from 10 healthy individuals served as control. Following stimulation with PEG-rHuMGDF 23+/-7% and 16+/-4% of control cells were CD42b positive after 5 and 7 d of cultures, respectively. In cultures of cells from MDS patients prior to treatment 8+/-2% and 7+/-3% of cells were CD42b+ on days 5 and 7. In the course of IFN alpha treatment cultures of all BM samples from these MDS patients revealed a significant reduction of MK precursor cells (3+/-2%, CD42b+, p=0.03 and 0.04). In conclusion, treatment with TFN alpha and ATRA did not result in improved megakaryocytopoiesis as assessed by in-vitro cultures. On the contrary, low-dose IFN alpha appears to suppress cell proliferation as well as MK development.

Administration, Oral↗

PubMed1996

Identification of genes induced by factor deprivation in hematopoietic cells undergoing apoptosis using gene-trap mutagenesis and site-specific recombination.

A strategy employing gene-trap mutagenesis and site-specific recombination (Cre/loxP) has been developed to isolate genes that are transcriptionally activated during programmed cell death. Interleukin-3 (IL-3)-dependent hematopoietic precursor cells (FDCP1) expressing a reporter plasmid that codes for herpes simplex virus-thymidine kinase, neomycin phosphotransferase, and murine IL-3 were transduced with a retroviral gene-trap vector carrying coding sequences for Cre-recombinase (Cre) in the U3 region. Activation of Cre expression from integrations into active genes resulted in a permanent switching between the selectable marker genes that converted the FDCP1 cells to factor independence. Selection for autonomous growth yielded recombinants in which Cre sequences in the U3 region were expressed from upstream cellular promoters. Because the expression of the marker genes is independent of the trapped cellular promoter, genes could be identified that were transiently induced by IL-3 withdrawal.

Animals↗

PubMed1996

Effects of Flt3 ligand and interleukin-7 on in vitro growth of acute lymphoblastic leukemia cells.

We investigated the effects of Flt3/Flk-2 ligand (FL) and interleukin-7 (IL-7) on DNA synthesis and proliferation of blast cells from patients with acute lymphoblastic leukemia (ALL). After 7 days of serum-free suspension culture of 19 samples, FL induced maximal DNA synthesis in two cases, whereas the combination of FL and IL-7 did so in another eight samples with a stimulation index (SI) >2. However, the number of viable cells after 7 days of liquid culture decreased in all but one sample. In this case of a pre-pre-B-ALL with a translocation t(4;11), FL induced dose-dependent proliferation (maximal 100 ng/mL) and cells stimulated with FL could be cultured for up to 4 weeks. A homogeneous population with 98% CD19-positive cells was detected before and after culture, and there was no evidence of nonleukemic cell proliferation as determined by immunophenotyping. The flt3 gene was transcribed in all seven cases studied by reverse-transcriptase polymerase chain reaction (RT-PCR). In the ALL cells responsive to FL, the expression of functional Flt3 receptors was confirmed by demonstrating FL-dependent tyrosine phosphorylation of Flt3. Furthermore, FL-dependent tyrosine phosphorylation of cellular proteins of estimated molecular weights of 70, 115, and 140 kD was detectable in these cells. These data demonstrate the functional heterogeneity of ALL samples and show that functional Flt3 receptors capable of mediating FL-dependent mitogenic signaling are expressed in a subset of ALL.

Adolescent↗