Biomedical subjects

M R Halie

Publications and source records attributed to M R Halie.

At least 37 records · Page 2Linked to original sources

PubMed1994

G(AnH)MTetra, a naturally occurring 1,6-anhydro muramyl dipeptide, induces granulocyte colony-stimulating factor expression in human monocytes: a molecular analysis.

N-Acetylglucosaminyl-1,6-anhydro-N-acetylmuramyl-L-alanyl-D-isoglutam yl-m- diaminopimelyl-D-alanine [G (Anh)MTetra], a naturally occurring breakdown product of peptidoglycan from bacterial cell walls, was studied for its ability to induce granulocyte colony-stimulating factor (G-CSF) mRNA and protein expression in human adherent monocytes. Resting monocytes did not express G-CSF mRNA or secrete G-CSF protein. In contrast, monocytes exposed to G(Anh)MTetra showed a dose-dependent increase in G-CSF mRNA accumulation, which correlates with the secretion of G-CSF protein. Maximal levels of G-CSF mRNA were reached within 2 h of activation. Expression of G-CSF was mediated by an increase in the stability of G-CSF transcripts rather than by an increase in the transcription rate of the G-CSF gene. Experiments with the protein synthesis inhibitor cycloheximide revealed that G(Anh)MTetra-induced G-CSF mRNA expression was independent of new protein synthesis. Furthermore, it was shown that the effect of G(Anh)MTetra was regulated by a protein kinase C-dependent pathway, whereas protein kinase A and tyrosine kinases were not involved. Finally, it was shown that G(Anh)MTetra also induced G-CSF mRNA expression in human endothelial cells. The data indicate that, besides lipopolysaccharide, other naturally occurring bacterial cell wall components are able to induce G-CSF expression in different hematopoietic cells.

Acetylmuramyl-Alanyl-Isoglutamine↗

PubMed1994

Comparison of imipenem versus cefuroxime plus tobramycin as empirical therapy for febrile granulocytopenic patients and efficacy of vancomycin and aztreonam in case of failure.

143 aplastic episodes with fever in 91 haematological patients with granulocytopenia were treated empirically in a randomized prospective study using either imipenem (Imi) or a combination of tobramycin and cefuroxime (T/C). Response after 72 h was significantly better in patients receiving Imi (44/75 vs 27/68, p < 0.05). This was seen especially in patients with bacteriologically proven infections where the isolated staphylococci and streptococci were more susceptible to Imi. In both groups, patients who failed to respond to the initial antibiotic therapy were given vancomycin and aztreonam (V/A). The response rate after another 72 h, measured using the same criteria as after the first 72 h, did not differ statistically between the groups. One patient in each study group died from the bacterial infection, both from Gram-positive bacteraemia. Duration of fever was significantly shorter in the Imi group (4 days vs 7 days, p < 0.04). Serum peak and trough concentrations of the antibiotics were comparable. Both regimens were well tolerated. Our results show that monotherapy with imipenem is superior to the combination of tobramycin and cefuroxime during the first 72 h of therapy and can be safely administered to neutropenic patients with predominantly Gram-positive infections. A combination of vancomycin and aztreonam, given when initial imipenem treatment has failed, was effective in only a few patients. Adjuvant glycopeptide therapy from the outset in the treatment of febrile granulocytopenic patients did not seem worthwhile.

Adolescent↗

PubMed1994

Interleukin-4-mediated inhibition of C-Fos mRNA expression: role of the lipoxygenase directed pathway.

Interleukin-4 inhibits several monocyte functions like A23187-induced expression of cytokines and c-fos and c-jun proto-oncogene mRNA expression. In an attempt to elucidate the mechanism by which this inhibitive effect is mediated, we compared the effect of IL-4 on A23187-induced c-fos and c-jun mRNA expression in conjunction with inhibitors that selectively inhibit the cyclooxygenase dependent (indomethacin) and lipoxygenase dependent (NDGA) pathway of arachidonic acid (AA) metabolism. NDGA inhibited A23187-induced c-fos mRNA expression by a similar magnitude as IL-4, whereas the effect of indomethacin was only minor. A23187-induced c-jun mRNA expression was not affected by indomethacin and only slightly inhibited by NDGA. These results indicate that in human monocytes c-fos mRNA expression is at least partly controlled by the lipoxygenase directed pathway of AA metabolism, whereas the cyclooxygenase dependent pathway is not involved in the regulation of proto-oncogene expression. This was supported by the finding that leukotriene B4 (LTB4) and 5'-hydroperoxyeicosatetraenoic acid (5'-HPTETE), which are two lipoxygenase metabolites, strongly induced c-fos mRNA, whereas c-jun mRNA expression was slightly affected. However, the inhibitive effect of IL-4 could not be ascribed to a reduced production of LTB4 suggesting that the mode of IL-4 action lies behind the conversion of AA to 5'-HPETE and LTB4.

Arachidonic Acid↗

PubMed1994

Interleukin-4 mRNA and protein in activated human T cells are enhanced by interleukin-7.

We studied the effect of the stroma-derived cytokine interleukin-7 (IL-7) on the expression of IL-4 in human T cells at mRNA and protein level. The results demonstrate that IL-7 did not induce IL-4 mRNA in resting T cells. However, concanavalin A (con A)-induced IL-4 mRNA expression was enhanced by costimulation with con A plus IL-7. Nuclear run-on analysis revealed that IL-7 did not affect the transcription rate of the IL-4 gene. The half-life of con A-induced IL-4 transcripts, however, was increased upon con A plus IL-7 treatment, indicating that the effect of IL-7 is mediated at posttranscriptional level. In accordance with the mRNA results, IL-4 protein was not detected in supernatants of unstimulated T cells or T cells exposed to IL-7. In contrast, IL-7 augmented the con A-induced secretion of IL-4 protein significantly. In addition, it was noticed that anti-IL-1 beta and anti-tumor necrosis factor-alpha (anti-TNF-alpha) did not abolish the effect of IL-7 on the con A-induced IL-4 secretion, indicating that the IL-7 effect is not mediated by the release of these cytokines. These results indicate that a stroma-derived factor can affect IL-4 expression in activated human T cells.

Cells, Cultured↗

PubMed1993

The course of preexistent immune abnormalities in HIV negative haemophiliacs treated for two years with a monoclonal purified factor VIII concentrate.

The administration of factor VIII concentrates has been associated with immune abnormalities in patients with severe haemophilia A, even in the absence of HIV infection. The effects of a monoclonal purified factor VIII concentrate, Hemofil M (Baxter), on preexistent immune abnormalities were assessed over a 2 year period in 22 HIV negative haemophiliacs. They were treated previously with other concentrates, and received Hemofil T from 1983 to 1988. No HIV infection was demonstrated. No serologic evidence for other viral (re)-infections was seen. A decrease of HLA-DR expression on non-B lymphocytes in the first year (P = 0.026), and a decrease of T4-T8 ratio over the 2 years were found (P = 0.0016). Skin tests were non-contributive. The decrease in HLA-DR expression is suggestive for an improved immune function, possibly due to a reduced content of protein or virus in this concentrate.

Adolescent↗

PubMed1993

IL-7 enhances the expression of IL-3 and granulocyte-macrophage-CSF mRNA in activated human T cells by post-transcriptional mechanisms.

The stromal derived growth factor IL-7 was studied for its ability to modulate cytokine expression in human T cells. IL-7 alone did not induce IL-3 or granulocyte-macrophage-CSF (GM-CSF) mRNA. However, IL-7 enhanced the Con A-induced IL-3 and GM-CSF mRNA accumulation in a dose-dependent way. mRNA stability studies revealed that the effect of IL-7 was caused by post-transcriptional stabilization of the IL-3 and GM-CSF transcripts. Upon Con A treatment, the IL-3 and GM-CSF mRNA decayed with a t1/2 of approximately 90 and 50 min, respectively. Costimulation with Con A plus IL-7 stabilized both transcripts to t1/2 of greater than 2 h for IL-3 mRNA and 90 min for GM-CSF mRNA. Using nuclear run-on assays, we showed that the transcription rate of both genes was not affected by IL-7. Furthermore, it appeared that the effect of IL-7 was independent on protein synthesis, because cycloheximide did not abolish the promotive effect of IL-7. Finally, it was shown that in accordance with the mRNA results IL-7 enhanced the secretion of GM-CSF protein in Con A-activated T cells. After 12 h of stimulation T cells cultured in the presence of Con A secreted 575 +/- 309 pg GM-CSF/ml (x +/- SD, n = 5), which increased to 1425 +/- 758 pg/ml in the presence of Con A plus IL-7 (p < 0.01). In summary, these data demonstrate that IL-7 augments the expression and secretion of CSF in activated human T cells.

Gene Expression Regulation↗

PubMed1993

Interleukin-4 inhibits the lipopolysaccharide-induced expression of c-jun and c-fos messenger RNA and activator protein-1 binding activity in human monocytes.

We studied the effect of interleukin-4 (IL-4) on the lipopolysaccharide (LPS) induction of two immediate early genes c-fos and c-jun. These genes encode proteins that form the dimeric complex activator protein-1 (AP-1), which is active as a transcriptional factor. Maximal accumulation of either c-fos and c-jun messenger RNA (mRNA) occurred 30 minutes after LPS addition. When cells were treated with IL-4 for 5 hours before LPS activation, both the c-fos and the c-jun mRNA expression was decreased. The inhibition of c-fos and c-jun expression by IL-4 in LPS-treated cells was shown to be due to a lower transcription rate of the c-fos and c-jun genes. IL-4 did not affect the stability of the c-fos and c-jun transcripts. Finally, using electrophoretic mobility shift assays, evidence was obtained that IL-4 inhibits LPS-induced expression of AP-1 protein. These data indicate that IL-4 suppresses the induction of transcription factors in human activated monocytes.

Base Sequence↗

PubMed1993

Transcriptional and posttranscriptional regulation of the interleukin-4 and interleukin-3 genes in human T cells.

Human T cells were studied with regard to the regulation of interleukin-4 (IL-4) and IL-3 gene expression. IL-4 and IL-3 mRNA were undetectable in unstimulated T cells. On activation with the lectin concanavalin A (Con A), both IL-4 and IL-3 mRNA were expressed. Accumulation of IL-4 mRNA peaked after 6 to 12 hours, whereas IL-3 mRNA levels peaked after 3 to 6 hours of stimulation with Con A. Nuclear run-on assays showed a low constitutive transcription for both genes. The transcription rates were increased by Con A resulting in a peak for IL-4 after 1 hour (30% increase) and for IL-3 after 3 hours (40% increase) of Con A treatment. mRNA stability studies demonstrated that on activation with Con A both messages decayed with a half-life of approximately 90 minutes. No IL-4 or IL-3 mRNA expression was induced by the protein kinase C activator phorbol myristate acetate (PMA). However, PMA augmented the Con A-induced IL-4 and IL-3 mRNA accumulation. This was shown to be mediated at posttranscriptional level by a large increase in the stability of both messages (t 1/2 > 3 hours). The transcription rate of both genes was also enhanced by Con A+PMA and reached peak levels for IL-4 after 1 hour (90% increase) and for IL-3 after 3 hours (70% increase) of stimulation. Furthermore, it appeared that the induction of IL-4 mRNA was dependent on protein synthesis because cycloheximide (CHX) blocked the Con A- and Con A+PMA-induced expression of IL-4 mRNA. In contrast, CHX inhibited, but failed to completely block, the Con A- and Con A+PMA-induced IL-3 mRNA expression, whereas the expression of both genes was completely blocked by cyclosporine A. With regard to the secretion of IL-4 protein it was shown that it closely follows the accumulation of IL-4 mRNA. Taken together, the data show that expression of the IL-4 and IL-3 genes in human T cells is controlled by different activation pathways that affect the gene regulation at transcriptional and posttranscriptional levels.

Blotting, Northern↗

PubMed1993

Changes of buoyant density during the S-phase of the cell cycle. Direct evidence demonstrated in acute myeloid leukemia by flowcytometry.

Studies with synchronized or exponentially growing bacteria and mammalian cell lines are not able to demonstrate small changes in buoyant density during the cell cycle. Flowcytometric analysis of density separated acute myeloid leukemia cells, a system not dependent on time-related variables, shows that the cellular buoyant density increases slightly with up to 0.008 g/ml during the S-phase, at least in cryo-preserved cells used in this study. This contrasts with the generally accepted belief that S-phase cells have a lower or constant buoyant density. A practical implication is that separation of cell (sub)populations based on differences in buoyant density could be flawed to the extent that these populations contain S-phase cells.

DNA↗

PubMed1993

The clinical expression of hereditary protein C and protein S deficiency: a relation to clinical thrombotic risk factors and to levels of protein C and protein S?

We investigated 103 first-degree relatives of 13 unrelated protein C or protein S deficient patients to assess the role of additional thrombotic risk factors and of protein C and protein S levels in the clinical expression of hereditary protein C and protein S deficiency. Fifty-seven relatives were protein C (37) or protein S deficient (20). Thromboembolic events occurred in 30% of protein C deficient and in 35% of protein S deficient persons, compared with 3% and 0% in their normal controls respectively (P < 0.05). In protein C deficient persons, the median thromboembolic event-free survival was 55 years, while in protein S deficiency this interval was 33 years (P = 0.047). In the protein C deficient group 64% of the initial events occurred spontaneously, as did 71% in the protein S deficient group. Recurrent thromboembolic events were more often associated with concomitant risk factors than the initial events: 64% and 50% in persons with protein C or protein S deficiency respectively. These findings suggest a substantial role for these risk factors in triggering thromboembolic events in deficient persons. Protein C antigen and protein S antigen levels were similar in symptomatic and asymptomatic deficient persons. Total, but not free, protein S antigen levels were significantly higher in symptomatic protein C deficient persons, as were protein C antigen and activity levels in symptomatic protein S deficient ones. The clinical implication of this finding is not yet clear.

Adolescent↗

PubMed1993

Interferon-gamma enhances the LPS-induced G-CSF gene expression in human adherent monocytes, which is regulated at transcriptional and posttranscriptional levels.

Human adherent monocytes were studied with regard to the expression of granulocyte colony-stimulating factor (G-CSF) at mRNA and protein levels in response to lipopolysaccharide (LPS) and gamma-interferon (IFN-gamma) stimulation. Monocytes did not express G-CSF transcripts in response to IFN-gamma treatment. In contrast, monocytes exposed to IFN-gamma plus LPS showed a dose-dependent increase in G-CSF mRNA accumulation and protein secretion compared to LPS-stimulated monocytes. The augmented G-CSF mRNA expression in response to IFN-gamma plus LPS was the result of a slight increase in the G-CSF transcription rate (2.2-fold) and a more than 6-fold increase in the G-CSF mRNA half-life (20 minutes vs. > 120 minutes). In addition, it was shown that the effects of IFN-gamma on LPS-induced G-CSF protein secretion could be mimicked by the calcium ionophore A23187, suggesting that the Ca(2+)-dependent pathway might be triggered after binding of the ligand to the receptor. Finally, it was observed that the potentiating effects of IFN-gamma on LPS-induced G-CSF secretion could be blocked by interleukin-4 (IL-4). These data indicate that two cytokines produced by activated T cells have opposite effects on G-CSF production by human activated monocytes.

Blotting, Northern↗

PubMed1993

Recombinant human mast cell growth factor supports erythroid colony formation in polycythemia vera in the presence and absence of erythropoietin and serum.

The effect of mast cell growth factor (MGF) was studied on erythropoietin (Epo)-dependent and Epo-independent ("spontaneous") erythroid colony formation in patients with polycythemia vera (PV). MGF stimulated both Epo-dependent and Epo-independent erythroid colony formation from PV peripheral blood progenitor cells in vitro at a dose similar to normal erythroid progenitor. In addition, evidence was obtained that the stimulating effect of MGF was a direct effect on the erythroid progenitor and independent of serum. Antibodies against interleukin-1 (IL-1), IL-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), and Epo could not abolish the enhancing effect of MGF. This was also supported by the finding that sorted CD34+ cells could be stimulated by MGF in the presence and absence of Epo. Finally, it was demonstrated that the spontaneous erythroid colony formation could not be ascribed to spontaneous release of MGF in the culture medium since anti-MGF did not affect the colony numbers. In conclusion, MGF has a direct stimulatory effect, independent of serum, on both Epo-dependent and Epo-independent erythroid colony formation in PV.

Antibodies↗

PubMed1992

Interleukin-4 (IL-4) receptor expression on human T cells is affected by different intracellular signaling pathways and by IL-4 at transcriptional and posttranscriptional level.

Interleukin-4 (IL-4) modulates the survival, proliferation, and differentiation of a variety of hematopoietic cells. The effects are mediated through a single class of high-affinity receptors for IL-4. To understand the biologic effects of IL-4 on human T cells, we studied the regulation of IL-4 receptor (IL-4R) gene expression. We showed that IL-4R mRNA accumulation in human T cells is enhanced fourfold after activation of different secondary signaling pathways by concanavalin A (Con A), phorbol myristate acetate (PMA), the calcium ionophore A23187, and combinations of these factors. This could be ascribed to an increase in the IL-4R transcription rate and to stabilization of IL-4R mRNA resulting in a half-life of 80 to 90 minutes (v 35 to 40 minutes in resting T cells). IL-4 did enhance the IL-4R mRNA accumulation by a factor 10, which was caused by an increase in the IL-4R transcription rate and prolonging the half-life of IL-4R transcripts to 140 to 160 minutes. Finally, it was shown that A23187 induced IL-4R mRNA expression is a protein synthesis-dependent process. In contrast, Con A-, PMA-, Con A + PMA-, and Con A + A23187-induced expression of IL-4R mRNA is protein-synthesis independent. Cyclosporine A inhibited the A23187- and Con A + A23187-induced IL-4R mRNA accumulation, whereas Con A-, PMA-, and Con A + PMA-induced IL-4R mRNA expression was not affected by this drug. These data indicate that expression of IL-4 receptors on human T cells can be modulated by different intracellular signaling pathways at both transcriptional and posttranscriptional levels.

Calcimycin↗

PubMed1992

The supportive effects of IL-7 on eosinophil progenitors from human bone marrow cells can be blocked by anti-IL-5.

Human rIL-7 was studied for its effects on myeloid and erythroid progenitors from human bone marrow cells. IL-7 did not support the granulocytic/monocytic or erythroid lineage but exclusively stimulated eosinophil colony formation (CFU-Eo) (4 +/- 3 vs 48 +/- 17 CFU-Eo/10(5) nonadherent fraction-non-T cell (NAF-NT) cells). This supportive effect was not mediated by T cells or monocytes because similar results were obtained with or without T cell or adherent depleted cell fractions. In addition, it was shown that CD34+ sorted cells could be stimulated by IL-7 (0 vs 15 +/- 9 CFU-Eo/3 x 10(3) CD34+ cells) Furthermore studies with IL-3 or granulocyte-macrophage CSF (GM-CSF) demonstrated an additive effect on the IL-7 supported colony formation. Finally, experiments were performed with anti-IL-3, anti-GM-CSF, anti-IL-1, and anti-IL-5 to exclude the possibility that IL-7 indirectly stimulated the eosinophil progenitor cell. Anti-GM-CSF, anti-IL-1, or anti-IL-3 did not influence the supportive effects of IL-7. However, anti-IL-5 did abolish the effects of IL-7 on the eosinophil colony formation (69 +/- 15 vs 3 +/- 2 CFU-Eo/10(5) NAF-NT, n = 3). Similar results were obtained with CD34+ sorted cells. Moreover, IL-5 mRNA expression could be demonstrated in IL-7-stimulated NAF-NT cells. These data suggest that the supportive effects of IL-7 on eosinophil precursors are mediated by the endogenous release of IL-5.

Antigens, CD↗

PubMed1992

Characteristics of red blood cell populations fractionated with a combination of counterflow centrifugation and Percoll separation.

Red blood cell (RBC) fractions were studied after separation of whole blood by means of counterflow centrifugation, Percoll column (Pharmacia, Uppsala, Sweden), and a combination of both separation techniques. Mean corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), mean corpuscular hemoglobin (MCH), and hemoglobin A1c (HbA1c) were measured in each fraction. From the results it was obvious that the combination of both techniques was the best separation technique of these three. MCV had a good correlation with cell age as measured with HbA1c concentration gradient; MCH and MCHC less so. MCV and MCH decreased in parallel to an increase in HbA1c. MCHC increased with increasing HbA1c. From these data it is concluded that there is a steadily ongoing loss of cellular hemoglobin and proportionally more cellular water during the life of the RBC.

Cell Separation↗

PubMed1992

Evaluation of elutriated single donor platelets collected and stored in a closed system.

Single donor platelets (SDPC) were collected by the elutriation technique in a closed-system integrated with large storage containers. Seven runs of SDPC were stored in a 1.5 liter polyvinyl-chloride trimellitate (PVC-TOTM) storage container, making the ratio of platelet concentrate volume to container volume 1:4.5. An equal volume of pooled multiple donor platelet concentrates (MDPC) was stored in parallel under the same conditions. All haematological data were comparable for both products, except for the degree of leukocyte contamination (5-fold increase in the pool). Under these conditions, the functional, morphological, and metabolic characteristics of elutriated platelets throughout 7-day storage were superior to those of pooled platelets. Although the platelet count was not significantly different in both types of concentrates, the mean pH of pooled MDPC fell to 6.0 on day 5 of storage. Leukocytes were shown to contribute to this pH fall. The extent of cell damage, however, as evidenced by LDH leakage (42.7 LDH units/10(11) platelets/day by differential centrifugation, compared to 5.3 units by elutriation) could not be explained solely on the basis of the leukocyte effect. This indicated that the processing method itself influences the platelet quality. By increasing the surface/volume ratio of SDPC, the initial pH of 7.1 was well maintained throughout storage, platelet metabolic rate was slowed, and the function and ultrastructure improved significantly.

Blood Donors↗

PubMed1992

Stabilization of standard platelet concentrates and minimization of the platelet storage lesion by a prostacyclin analogue.

Platelet concentrates were pretreated with a stable synthetic prostacyclin analogue (Iloprost) at two different concentrations before the second centrifugation step (pelleting step) of preparation. This resulted in loss of platelet sensitivity to aggregating agents. To mimic the situation after transfusion and to assess the reversibility of platelet inhibition, platelets were washed during and after storage and resuspended in fresh-frozen autologous plasma. The Iloprost-treated and washed platelets exhibited an increased sensitivity to the aggregating agents, compared with the control platelets (p less than 0.01). Post-storage recovery of the synergistic aggregation was more than 80% of prestorage aggregation. Beta-thromboglobulin (beta TG) release and thromboxane B2 (TXB2) formation were significantly inhibited in Iloprost-treated platelets (p less than 0.01). After the second centrifugation step, beta TG release was 0.7% +/- 0.3%, compared with 2.7% +/- 0.9% for the controls. TXB2 was 99 +/- 91 pg/ml, compared with 495 +/- 356 pg/ml for the controls. Platelet morphology and ultrastructure were well preserved during 5-day storage. In addition, Iloprost exerted a cytoprotective effect, as evidenced by the significant reduction in lactate dehydrogenase leakage. Post-storage LDH was 378 +/- 159 and 415 +/- 239 U/l respectively by the two Iloprost concentrations, compared with 1180 +/- 937 U/l for the control platelets. The inhibitory and cytoprotective effects of Iloprost were sustained throughout storage, in contrast to the effect of PGE1 (Prostin) which was limited to the early phase of storage.

Alprostadil↗

PubMed1992

Influence of high versus low intestinal concentration of gram-negative bacteria and endotoxin on the susceptibility of murine myelopoiesis in bone marrow and spleen to cytostatic treatment with Ara-C.

The haematopoietic recovery after i.v. cytarabine was studied in C3H/Law mice as a measure for stem cell susceptibility in relation to the intestinal Gram-negative bacteria (GNB) and endotoxin. Reduction or elevation of GNB and endotoxin was induced by either polymyxin or bacitracin, both non-absorbable antibiotics. Bacitracin caused less suppression of the splenic cellularity after cytarabine, and an advancement of the recovery of femoral nucleated cells. The femoral recovery of CFU-GM exhibited a biphasic pattern. The speed and height of the rebound increase of CFU-GMs were significantly affected by the antibiotics. Thus, (modulation of) the murine intestinal microflora influences the haematopoietic recovery after cytostatic drugs. The mechanisms involved are complex; intestinal endotoxin seems to play a role.

Animals↗