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Biomedical subjects

W Jelkmann

Publications and source records attributed to W Jelkmann.

At least 37 records · Page 2Linked to original sources
PubMed2001

Hepatic thrombopoietin mRNA is increased in acute inflammation.

The plasma concentration of thrombopoietin (TPO) in general is inversely related to the mass of platelets and megakaryocytes. However, reactive thrombocytosis of inflammatory disease is accompanied by elevated TPO levels. To investigate whether the rate of TPO mRNA expression is altered during acute inflammation, rats were injected with bacterial lipopolysaccharide (LPS). After 6 h, total RNA from liver and kidney was reverse transcribed and analyzed by competitive PCR for TPO and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). LPS-treated rats showed a significant increase in hepatic TPO mRNA concentration. The ratio of TPO to GAPDH mRNA was 3.5 +/- 0.6% in the livers of control rats and 8.3 +/- 2.0% in the livers of LPS-treated rats (mean +/- SD). Thus, reactive thrombocytosis of inflammatory disease might result from an increase in hepatic TPO production. Since platelets are involved in the immune reaction, reactive thrombocytosis may be a mechanism of host defense.

Acute Disease↗
PubMed2000

Nucleotide sequence and genome organisation of cherry mottle leaf virus and its relationship to members of the Trichovirus genus.

The nucleotide sequence of cherry mottle leaf virus (CMLV) was determined and compared to sequences of a number of plant viruses including the type member of the Trichovirus genus (apple chlorotic leafspot virus, ACLSV), and members of the Vitivirus genus including grapevine virus B, (GVB). The CMLV genome was determined to consist of 8003 nt excluding the poly(A) tail at the 3' end of the genome. The overall A + U content of CMLV genomic RNA was 59.1%, which is similar to ACLSV, but significantly different from GVB. Four putative open reading frames were identified (ORFs 1, 2, 3, and 4) encoding proteins of M(r) 215.8 kDa, 47 kDa, 21.6 kDa, and 15.3 kDa, respectively. This differs from ACLSV which has 3 ORFS, and GVB which has 5 ORFs. Protein database searches showed no matches of CMLV ORF4 with ACLSV sequences, but found similarities between ORF4 of CMLV and ORF5 of GVB, suggesting that this may be a nucleic acid-binding protein. CMLV and ACLSV formed a common virus clade in phylogenetic analysis of the coat protein amino acid sequence and except for CMLV's ORF4, these viruses show high levels of similarity throughout the genome. CMLV appears to be a member of the Trichovirus genus.

Amino Acid Sequence↗
PubMed2000

Increase in immune activation, vascular endothelial growth factor and erythropoietin after an ultramarathon run at moderate altitude.

The present study was performed to investigate the effects of exhaustive long lasting exercise at moderate altitude on the time course of serum immunomodulatory peptides, vascular endothelial growth factor (VEGF) and serum erythropoietin (EPO). Thirteen well trained runners participated at the Swiss Alpine Marathon of Davos (distance 67 km, altitude difference 2300 m). Interleukin-6 was significantly elevated in the first 2h after the run. In contrast, tumor necrosis factor-alpha and both soluble tumor necrosis factor-a receptors I and II were increased after exercise termination and showed sustained serum concentrations the following days. Neopterin, a serum marker for the activation of the cellular immune system, was increased until day two after the run. Immediately after the run VEGF was significantly elevated and further increased 2.4-fold until day five post exercise (p = 0.005). EPO was also increased after exercise but reached its maximum 2 h after the run (2-fold increase; p = 0.004) and decreased thereafter. The main findings of our study are that prolonged strenuous exercise at moderate altitude induced a significant long lasting increase in serum VEGF and EPO which was accompanied by an activation of the immune system.

Adult↗
PubMed2000

Low platelet count and elevated serum thrombopoietin after severe trauma.

Platelet count is regularly low in patients after multiple trauma, mainly due to blood loss and dilution. Thrombopoietin (TPO) is the main regulator of the circulating platelet mass. Under several clinical conditions an inverse correlation between TPO and the circulating platelet mass was reported. Since platelets bind and internalize TPO, a platelet-dependent regulation of TPO was suggested. Thus, acute blood loss should be accompanied by elevated TPO. We measured serum TPO, platelets, interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) in 17 multiple traumatized victims. Blood was collected within 12 h after trauma as well as in the morning of days 2, 4, 6 and 9 after admission at the intensive care unit. Platelet count was low at admission and remained low until day 4. Thereafter platelets increased until day 9. TPO nearly doubled within the first 2 d, reaching its maximum on day 6. IL-6 was initially very high and steadily decreased until day 9. VEGF increased 3-fold during the 9 d. Statistically significant correlations of TPO were found with platelets and IL-6, but not with VEGF. In multiple traumatized patients low platelet count is followed by a rapid increase in serum TPO. This fits into the concept of a feedback regulation between circulating TPO and platelet mass.

Adult↗
PubMed2000

Hypoxia and interleukin-1beta stimulate vascular endothelial growth factor production in human proximal tubular cells.

BACKGROUND: Vascular endothelial growth factor (VEGF) promotes angiogenesis and inflammatory reactions. VEGF mRNA is detectable in the proximal tubules of inflamed kidneys but not in normals. In other organs VEGF gene expression is induced by hypoxia and cytokines such as interleukin 1 (IL-1). To identify the cellular mechanisms in control of tubular VEGF production, we studied effects of hypoxia and IL-1beta in VEGF mRNA levels, VEGF secretion, and activity of the hypoxia-inducible dimeric transcription factor 1 (HIF-1alpha/beta) in human proximal tubular epithelial cells (PTECs) in primary culture. METHODS: PTECs were grown in monolayers from human kidneys. Hypoxia was induced by incubation at 3% O2. VEGF mRNA was quantitated by competitive polymerase chain reaction following reverse transcription. VEGF was measured by enzyme-linked immunoassay. HIF-1alpha was demonstrated by Western blot analysis and HIF-1 DNA binding by gel shift assay. RESULTS: Significant amounts of VEGF mRNA and VEGF protein were measured in PTEC extracts and culture media, respectively. Stimulation of VEGF synthesis at low O2 tension and following IL-1beta treatment was detectable at the protein level only. Nuclear HIF-1alpha protein levels and HIF-1 binding to DNA were also increased under these conditions. CONCLUSIONS: PTECs in culture produce VEGF. One mechanism of induction appears to be increased DNA binding of HIF-1 to hypoxia-responsive elements in the VEGF gene promoter. In inflammatory diseases of the kidney, tubular cell-derived VEGF may contribute to microvascular leakage and monocyte extravasation.

Animals↗
PubMed2000

Interleukin-6 increases thrombopoietin production in human hepatoma cells HepG2 and Hep3B.

The concentration of circulating thrombopoietin (TPO) is relatively high in patients with thrombocytosis reactive to inflammatory diseases. We investigated whether immunomodulatory cytokines stimulate TPO synthesis in cultured human hepatoma cells (lines HepG2 and Hep3B), renal proximal tubular cells, and bone marrow fibroblasts. The effects of interleukins (IL) IL-1beta, IL-6, and IL-11 and of tumor necrosis factor-a (TNF-alpha) on the rate of TPO secretion were measured by ELISA. TPO mRNA levels were quantitated by competitive reverse transcription PCR. HepG2 and Hep3B cells produced significant amounts of TPO mRNA and TPO protein. Renal tubular cells synthesized less TPO, and in bone marrow fibroblasts, neither TPO mRNA nor TPO protein was detected. Only IL-6 affected TPO protein secretion, causing a 1.5-fold stimulation in HepG2 and Hep3B cells in 24-h incubation periods. The TPO mRNA content in these cells was doubled by IL-6 after 2, 6, or 24 h of stimulation. Thus, IL-6 could cause thrombocytosis in inflammatory disease partly by increasing hepatic TPO production.

Base Sequence↗
PubMed2000

Tumor necrosis factor-alpha and TNF-beta inhibit clonogenicity of mobilized human hematopoietic progenitors.

We studied the effect of tumor necrosis factors (TNF-alpha and TNF-beta) on the clonogenicity of peripheral blood progenitor cells (PBPC) mobilized by granulocyte colony-stimulating factor (G-CSF). The cells were obtained by nine leukaphereses in patients with malignancies undergoing peripheral blood stem cell transplantation (PBSCT). Flow cytometry was performed to evaluate the number of CD34+ cells in peripheral blood and leukapheresis products. PBPC were grown in semisolid medium supplemented with human growth factors in the absence or presence of TNF at concentrations ranging from 0.1 to 10 ng/ml. Colonies were scored on day 14. TNF-alpha and TNF-beta suppressed colony formation in all cases studied. TNF-alpha inhibited the growth of colony-forming units-granulocyte-macrophage (CFU-GM) at 1 and 10 ng/ml and burst-forming units-erythroid (BFU-E) at 10 ng/ml (p < 0.05), and TNF-beta inhibited the growth of CFU-GM and BFU-E at 1 and 10 ng/ml (p < 0.05). Thus, G-CSF-mobilized hematopoietic PBPC are highly sensitive to both TNF-alpha and TNF-beta. This finding should be taken in account when PBPC are handled ex vivo.

Cell Culture Techniques↗
PubMed2000

Detection of beet yellows closterovirus methyltransferase-like and helicase-like proteins in vivo using monoclonal antibodies.

In the positive-stranded RNA genome of beet yellows closterovirus (BYV), the 5'-terminal ORF 1a encodes a 295 kDa polyprotein with the domains of papain-like cysteine proteinase, methyltransferase (MT) and helicase (HEL), whereas ORF 1b encodes an RNA-dependent RNA polymerase. Eleven and five hybridoma cell lines secreting monoclonal antibodies (MAbs) were derived from mice injected with the bacterially expressed fragments of the BYV 1a product encompassing the MT and HEL domains, respectively. On immunoblots of protein from BYV-infected Tetragonia expansa plants, four MAbs against the MT recognized a approximately 63 kDa protein, and two MAbs against the HEL recognized a approximately 100 kDa protein. Both the methyltransferase-like protein and the helicase-like protein were found mainly in the fractions of large organelles (P1) and membranes (P30) of the infected plants. These data clearly indicate that (i) the BYV methyltransferase-like and helicase-like proteins, like other related viral enzymes, are associated with membrane compartments in cells, and (ii) the 1a protein, apart from the cleavage by the leader papain-like proteinase that is expected to produce the 66 kDa and 229 kDa fragments, undergoes additional processing by a virus-encoded or cellular proteinase.

Amino Acid Sequence↗
PubMed2000

Use of recombinant human erythropoietin as an antianemic and performance enhancing drug.

The glycoprotein hormone erythropoietin is an essential viability and growth factor for the erythrocytic progenitors in the bone marrow. Tissue hypoxia is the main stimulus for the synthesis of the hormone in the kidneys and the liver. Endogenous erythropoietin and recombinant human erythropoietin (rHu-EPO) are similar with respect to their biological and chemical properties except for some microheterogeneities in their 4 carbohydrate chains. Generic products and alternatives to rHu-EPO are in development. Renal anemia can be corrected by rHu-EPO in a dose-dependent and predictable way without major side effects apart from a possible increase in arterial blood pressure. The optimal target hematocrit still needs to be defined. There are rare reports of antibody formation towards rHu-EPO in humans. Patients suffering from non-renal anemias may also benefit from the prescription of rHu-EPO. The drug has been approved for treatment of tumor patients with platinum-induced anemia. The cost-effectiveness and medical justification of the administration of rHu-EPO in tumor patients with respect to its positive effects on tumor oxygenation, tumor growth inhibition and support of chemo- and radiotherapy is still a matter of debate. In surgical patients, the pharmacological application of rHu-EPO can increase the yield of blood units in autologous blood donation programs and lower the severity and duration of postoperative anemia, if applicated some days prior to surgery. While rHu-EPO is a godsend in medical practice, its abuse as an performance enhancing drug by athletes in endurance sports is an unethical and potentially dangerous procedure. Unequivocal methods for detection of rHu-EPO doping still need to be established.

Anemia↗
PubMed1999

Interleukin-1beta and tumor necrosis factor-alpha stimulate DNA binding of hypoxia-inducible factor-1.

The rate of transcription of several genes encoding proteins involved in O(2) and energy homeostasis is controlled by hypoxia-inducible factor-1 (HIF-1), a heterodimeric DNA binding complex composed of alpha and beta subunits. HIF-1 is considered the primary trans-acting factor for the erythropoietin (EPO) and vascular endothelial growth factor (VEGF) genes. Since EPO gene expression is inhibited by the proinflammatory cytokines interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha), while no such effect has been reported with respect to the VEGF gene, we investigated the effects of IL-1beta and TNF-alpha on the activation of the HIF-1 DNA-binding complex and the amount of HIF-1alpha protein in human hepatoma cells in culture. Under normoxic conditions, both cytokines caused a moderate activation of HIF-1 DNA binding. In hypoxia, cytokines strongly increased HIF-1 activity compared with the effect of hypoxia alone. Only IL-1beta increased HIF-1alpha protein levels. In transient transfection experiments, HIF-1-driven reporter gene expression was augmented by cytokines only under hypoxic conditions. In contrast to their effect on EPO synthesis, neither IL-1beta nor TNF-alpha decreased VEGF production. The mRNA levels of HIF-1alpha and VEGF were unaffected. Thus, cytokine-induced inhibition of EPO production is not mediated by impairment of HIF-1 function. We propose that HIF-1 may be involved in modulating gene expression during inflammation.

Animals↗
PubMed1999

Expression of the thrombopoietin gene in human fetal and neonatal tissues.

Thrombopoietin (TPO) regulates megakaryopoiesis and platelet production. In the adult, TPO is mainly produced by the liver and the kidneys. This study focuses on fetal and neonatal TPO mRNA expression. In 26 human fetuses and preterm neonates, samples from liver, kidney, spleen, lung, and bone marrow were extracted for total RNA. We measured platelet counts, TPO serum concentrations by enzyme-linked immunosorbent assay, and TPO mRNA contents by reverse transcription/competitive polymerase chain reaction. TPO mRNA concentrations per microgram total RNA were similar in liver, spleen, and bone marrow, slightly lower in kidney, and significantly lower in lung. When related to gram tissue, TPO mRNA levels were highest in the liver. Considering the total amount of TPO mRNA produced in liver, kidney, and spleen, the liver accounted for 95.3%. No correlations between TPO mRNA expression and serum TPO concentration, blood platelet count, or gestational age were observed. In conclusion, the liver is the primary site of TPO gene expression in human fetuses and neonates. The spleen may contribute to TPO production during fetal life. Like in the adult, TPO mRNA is expressed in fetal bone marrow.

Adult↗
PubMed1999

Evidence against a major role for Ca2+ in hypoxia-induced gene expression in human hepatoma cells (Hep3B).

1. The human hepatoma cell line Hep3B is a widely used model for studies of hypoxia-related gene expression. Cytosolic free calcium concentration ([Ca2+]i) has been implicated in cellular oxygen-sensing processes. We investigated whether calcium ions have a significant impact on the production of erythropoietin (EPO) and vascular endothelial growth factor (VEGF). 2. We found that the calcium ionophore ionomycin induced a rapid and sustained increase of [Ca2+]i while thapsigargin, an inhibitor of endoplasmic reticulum calcium ATPase, only caused a 20 % elevation of [Ca2+]i within 10 min after application. However, the calcium content of intracellular stores was considerably reduced by thapsigargin after an incubation period of 24 h. 3. Variations in [Ca2+]o did not result in altered EPO or VEGF secretion rates. Ionomycin decreased EPO production while the lowering of VEGF production was not statistically significant. In the presence of extracellular Ca2+ the membrane permeant calcium chelator BAPTA-AM stimulated the production of EPO (P < 0.05) but not of VEGF while EGTA-AM, a closely related agent, affected neither EPO nor VEGF formation under these conditions. Incubation with thapsigargin resulted in decreased EPO synthesis (P < 0.05) but stimulated VEGF secretion (P < 0.05). 4. In the absence of extracellular calcium, EGTA-AM led to an accumulation of hypoxia-inducible factor-1alpha (HIF-1alpha). This treatment significantly stimulated VEGF synthesis but also decreased EPO secretion (P < 0.05). 5. Our data suggest that the calcium transient and the cytosolic Ca2+ concentration do not play a key role in hypoxia-induced EPO and VEGF production in Hep3B cells.

Blotting, Northern↗
PubMed1999

Hepatic thrombopoietin mRNA levels in acute and chronic liver failure of childhood.

The liver is the main production site of the hormone thrombopoietin (TPO), the major regulator of megakaryopoiesis. To investigate the role of an impaired TPO gene expression in the pathogenesis of thrombocytopenia in pediatric patients suffering from liver failure, we measured hepatic TPO mRNA in children with acute or chronic end-stage liver disease undergoing orthotopic liver transplantation. Tissue samples for RNA extraction were obtained from 12 children with compensated cirrhosis (CC), 22 children with decompensated cirrhosis (DC), and 9 children with acute liver failure (ALF). TPO mRNA was quantitated by competitive polymerase chain reaction (PCR), following reverse transcription (RT). Furthermore, in 9 children with ALF, serum TPO levels were measured by enzyme-linked immunosorbent assay before and 10 to 14 days after liver transplantation. The hepatic TPO mRNA concentration was highest in children with CC (median, 50.9 amol/micrograms RNA). This value was significantly reduced in children with DC (30.2 amol/micrograms RNA) or ALF (13.8 amol/micrograms RNA). Children with ALF (139 cells/nL) or DC (200 cells/nL) had lower platelet counts than children with CC (368 cells/nL). The serum TPO concentration increased from a median of 156 pg/mL in patients with ALF to 547 pg/mL after liver transplantation. These results show that the thrombocytopenia in children with liver failure is associated with reduced hepatic TPO mRNA levels. It remains to be investigated whether the serum TPO level and platelet counts are markers for the severity of liver damage that may serve as a prognostic indicator.

Adolescent↗
PubMed1999

Vascular endothelial growth factor in exercising humans under different environmental conditions.

It was the aim of this study to investigate the time course of changes in the serum concentrations of vascular endothelial growth factor (VEGF) during a regular survival training programme combined with food and fluid deprivation and during a high altitude marathon run. We studied soldiers of the Austrian Special Forces performing survival training at sea-level and marathon runners of the Posta Atletica who crossed the border between Chile and Argentina at altitudes up to 4722 m. Baseline data collected before the 1-week of survival training showed that the soldiers had normal VEGF [n = 8, 246.7 (SD 118.5) pg.ml(-1)] serum concentrations which remained unchanged during the course of the study. Before the high altitude marathon the subjects showed normal VEGF serum concentrations [178 (SD 84.5) pg.ml(-1)]. After the run VEGF concentrations were found to be significantly decreased [41.0 (SD 41.6) pg ml(-1), P < 0.01]. It was concluded that prolonged physical stress during normobaric-normoxia did not alter the VEGF concentrations whereas during severe hypobaric-hypoxia decreased VEGF serum concentrations were measured, at least temporarily, after prolonged physical exercise which might have been due to changes in production, release, removal and/or binding of circulating VEGF.

Adolescent↗
PubMed1999

Tumor necrosis factor p55 receptor (TNF-RI) mediates the in vitro inhibition of hepatic erythropoietin production.

Tumor necrosis factor alpha (TNFalpha) is thought to contribute to the blunted erythropoietin (Epo) production in inflammatory diseases. The present study was carried out to find out as to whether the 55 kD (TNF-RI) or the 75 kD (TNF-RII) receptor is responsible for the TNFalpha-induced inhibition of hepatic Epo synthesis. When the effects of two receptor-specific mutants were compared, only the TNF-RI-specific isoform proved to suppress the formation of immunoreactive Epo in the human hepatoma cell lines HepG2 and Hep3B, similar to the effect of wild-type TNFalpha. Anti-TNFalpha antibody restored Epo production in TNFalpha- or TNF-RI mutant-treated cultures. By gel shift assay NF-kappaB binding to DNA was demonstrated following the addition of TNFalpha or TNF-RI-specific mutant to HepG2 cells, while the TNF-RII-specific mutant was ineffective. Finally, immunoreactive TNF-RI, but not TNF-RII, fragments were measurable in cell culture supernatants. Taken together, these results suggest that the inhibition of hepatic Epo production by TNFalpha is mediated by TNF-RI signaling.

Antigens, CD↗
PubMed1999

Elevated serum thrombopoietin and interleukin-6 concentrations in thrombocytosis associated with inflammatory bowel disease.

Reactive thrombocytosis is a typical feature in inflammatory bowel disease (IBD). The question arose as to whether the normal negative feedback regulation of the concentration of thrombopoietin (TPO) in blood was altered in IBD patients. We measured serum immunoreactive TPO in 30 patients with active IBD, 29 patients with inactive IBD, and 56 healthy controls. The results were related to platelet and leukocyte counts and to the serum concentration of interleukin 6 (IL-6). Patients with active IBD exhibited significantly increased TPO levels (medians 112 pg/ml vs. 90 pg/ml in controls, p < 0.05) in association with thrombocytosis (428 platelets/nl blood vs. 241 platelets/nl blood in controls), leukocytosis, and increased IL-6 levels (12.9 pg/ml vs. 2.5 pg/nl in controls). In patients with inactive IBD, only platelets (322/nl) and leukocytes were above normal. Although the observation of increased TPO and IL-6 levels provides an explanation for the occurrence of thrombocytosis in IBD, the pathogenetic mechanisms underlying the elevated TPO level still need to be identified.

Adolescent↗
PubMed1999

Serum erythropoietin and interleukin-6 levels in hemodialysis patients with hepatitis virus infection.

The influence of hepatitis B (HBV) and hepatitis C virus (HCV) infection on blood hemoglobin (Hb) and serum erythropoietin (Epo) and interleukin-6 (IL-6) concentrations was studied in 48 anemic patients on regular hemodialysis. They were grouped as follows: (I) 19 patients whose Hb values improved after infection (Hb > 85 g/L), (II) 10 patients with persisting anemia after infection (Hb < 75 g/L), and, without hepatitis virus markers (III) 8 patients with Hb > 85 g/L and (IV) 11 patients with Hb < 75 g/L. Serum immunoreactive Epo levels were significantly higher in group I (34.4+/-47.1 U/L) than in the other groups (II, 10.8+/-6.0; III, 7.9+/-3.2; IV, 8.4+/-4.3). Serum IL-6 was higher in group I than group III (7.7+/-7.8 pg/ml vs. 3.6+/-2.4; p = 0.05) but similar to the other groups. Hb levels in group I were maximal at the time of serum alanine aminotransferase normalization. Red cell production increases as a result of elevated circulating Epo during hepatic regeneration after HBV or HCV infection.

Anemia↗