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Biomedical subjects

E Holler

Publications and source records attributed to E Holler.

At least 91 records · Page 5Linked to original sources
PubMed1995

Critical involvement of transmembrane tumor necrosis factor-alpha in endothelial programmed cell death mediated by ionizing radiation and bacterial endotoxin.

In this report, we show that ionizing radiation (IR) at a clinically relevant dose (4 Gy) causes apoptosis in macrovascular and microvascular human endothelial cells. Treatment of irradiated cells with a low dose of bacterial endotoxin (LPS), similar to the levels observed in serum during endotoxemia, enhanced the rate of apoptosis, although LPS alone was unable to induce programmed cell death. The cytokine and endotoxin antagonist interleukin-10 (IL-10) reduced the rate of LPS + IR-induced apoptosis to levels obtained with irradiation alone. Using neutralizing antibodies against tumor necrosis factor-alpha (TNF), we could show crucial involvement of TNF in the LPS-mediated enhancement of IR-induced apoptosis, but not in the IR-induced apoptosis per se. However, further analysis strongly suggested the transmembrane form of TNF (mTNF), but not soluble TNF, to be accountable for the LPS-mediated cytotoxic effects. Studies with anatagonistic receptor specific antibodies clearly showed that TNF receptor type I (TR60) is essential and sufficient to elicit this effect. These findings are of potential clinical importance because they may disclose a relevant mechanism that leads to endothelial damage after radiotherapy or total body irradiation used for conditioning in bone marrow transplantation and that may thus contribute to transplant related complications, especially in association with endotoxemia or related inflammatory states.

Animals↗
PubMed1995

Large complexes of beta-poly(L-malate) with DNA polymerase alpha, histones, and other proteins in nuclei of growing plasmodia of Physarum polycephalum.

Of the various cell types in the life cycle of Physarum polycephalum, only the growing plasmodium contains the unusual polyester beta-poly(L-malate). The nuclei exhibit large complexes of this polymer with nuclear proteins, among them DNA polymerase alpha, histones, and HMG-like proteins. The complexes are indicated by the results of size exclusion chromatography and chemical cross-linking with 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride (EDC). After hydroxylaminolysis of the cross-linked polyester, the proteins are liberated and visualized on Western blots. The complexes of 1200-1400 kDa molecular mass exceed by far the size of free beta-poly(L-malate) and proteins. The observed variation in mass appears to be mainly a function of the kind and stoichiometry of the protein constituents and may explain the relatively high molecular mass in S phase and the low molecular mass during G2 phase of the mitotic cycle. The complexes are considerably stable at moderate ionic strength (100 mM KCl). Also, endogenous beta-poly(L-malate) does not exchange with added beta-[14C]poly(L-malate) during the lysis of the nuclei and the sample preparation. The complexes are dissociated at elevated concentrations of KCl, in the presence of spermine hydrochloride, or by treatment with DEAE/cellulose. Available evidence indicates that beta-poly(L-malate) may be involved in the maintenance of the plasmodial state of P. polycephalum.

Animals↗
PubMed1995

Graft-versus-leukemia effect of donor lymphocyte transfusions in marrow grafted patients.

The immune reactivity of allogeneic lymphocytes plays a major role in the control of leukemia after bone marrow transplantation. In patients with recurrent leukemia after marrow transplantation, chimerism and tolerance provide ideal conditions for adoptive immunotherapy with donor lymphocytes. We studied the effect of donor lymphocyte transfusions on acute and chronic leukemia in relapse after bone marrow transplantation. One hundred thirty-five patients with chronic myeloid leukemia (CML) (N = 84), acute myeloid leukemia (AML) (N = 23), acute lymphoblastic leukemia (ALL) (N = 22), myelodysplastic syndrome (MDS) (N = 5), and polycythemia vera with osteomyelofibrosis (PCV) (N = 1) were treated with transfusions of donor lymphocytes. Patients were monitored for response of leukemia, including in CML, the use of the polymerase chain reaction for bcr/abl mRNA transcripts and for the occurrence of graft-versus-host disease (GVHD) and myelosuppression. Complete remissions were induced by donor lymphocyte transfusions in 54 patients with CML (73%) and in the patient with PCV; complete remissions were also induced in five patients (29%) with AML and a patient with MDS. In contrast, ALL did not respond to adoptive immunotherapy with donor lymphocyte transfusions. Remissions were durable in patients treated for CML in chronic phase (probability of remission: 87% at 3 years). Lymphocyte transfusions were also given to 18 patients with ALL, AML, MDS, and transformed phase CML who were in remission after chemotherapy. These remissions were not durable. Fifty-two patients (41%) developed GVHD of grade 2 or more, and 41 patients (34%) showed signs of myelosuppression. Seventeen patients died without leukemia, 14 patients with GVHD and/or myelosuppression. Donor lymphocyte transfusions exert strong effects against myeloid forms of leukemia and induce durable remissions in CML.

Adolescent↗
PubMed1995

Modulation of acute graft-versus-host-disease after allogeneic bone marrow transplantation by tumor necrosis factor alpha (TNF alpha) release in the course of pretransplant conditioning: role of conditioning regimens and prophylactic application of a monoclonal antibody neutralizing human TNF alpha (MAK 195F).

Contribution of host-related cytokine release in the course of pretransplant conditioning to early tissue damage and induction of acute graft-versus-host disease (GVHD) after allogeneic bone marrow transplantation (BMT) has been shown in experimental models. We performed a clinical phase I/II trial applying a monoclonal antibody neutralizing human tumor necrosis alpha (TNF alpha) during pretransplant conditioning as additional prophylaxis in high-risk patients admitted to allogeneic BMT; TNF alpha serum levels and clinical courses in 21 patients receiving anti-TNF alpha prophylaxis were compared with data from 22 historical controls. Absence of significant release of TNF alpha in the period of busulphan (BUS) treatment, but significant induction of TNF alpha by total body irradiation (TBI) and cyclophosphamide (CY) conditioning were correlated with significantly earlier onset of acute GVHD in patients receiving TBI/CY regimens as compared with BUS/CY-treated patients. Prophylactic application of monoclonal anti-TNF alpha seemed to postpone onset of acute GVHD from day 15 to day 25 (P < .05) after TBI/CY and from day 33 to day 53 after BUS/CY (P < .10) conditioning. Application of monoclonal anti-TNF alpha in low and intermediate doses was safe and not associated with an increased incidence of infectious or hematologic complications. Thus, our data provide indirect and direct evidence for involvement of conditioning-related cytokine release in induction of early acute GVHD in the clinical setting and support further investigation of this novel approach in randomized trials.

Acute Disease↗
PubMed1995

Preparative synthesis of beta-L-malyl-coenzyme A assisted by malyl-coenzyme A synthetase from Pseudomonas AM1.

beta-L-Malyl-CoA was synthesized from L-malate, CoA, and ATP in the presence of catalytic amounts of L-malyl-CoA synthetase (thiokinase) from Pseudomona AM1, which had been 50-fold purified by protamine sulfate precipitation, ammonium sulfate precipitation, chromatography on DEAE-cellulose, and affinity chromatography on High Trap Blue in less than 2 days. The homogeneous enzyme was free of L-malyl-CoA lyase and showed 63% homology with succinyl-CoA synthetase from Thermus aquaticus in its N-terminal sequences. Yields of beta-L-[14C]malyl-CoA(1-10 mumol) were 70% before and 65% after purification in 0.1-0.5 mumol portions by high-performance liquid chromatography on a MN Nucleosil 100-7 C8 column. For most biochemical work, the product was partially purified with an overall 45% yield by chromatography on DEAE-Sephacel. The identity of the compound as beta-L-malyl-CoA was verified by chemical and enzymatic tests, and also in comparison with its chemically synthesized counterpart. The enzymatic synthesis, especially of radioactively labeled beta-L-malyl-CoA, is considerably faster, higher in yield, and less problematic than chemical synthesis.

Acyl Coenzyme A↗
PubMed1995

Genetic control of multiple sclerosis: increased production of lymphotoxin and tumor necrosis factor-alpha by HLA-DR2+ T cells.

Lymphotoxin (LT) and tumor necrosis factor-alpha (TNF-alpha) play an important role in the pathogenesis of multiple sclerosis (MS). MS is associated with the HLA-DR2, Dw2, DQ6 HLA class II haplotype. Because both LT and TNF-alpha are encoded in the HLA region, the HLA association of MS may be related to the production of these cytokines. To test this hypothesis, we investigated the production of LT, TNF-alpha, and interferon-gamma (IFN-gamma) by CD4+ T-cell lines (TCLs) specific for myelin basic protein (MBP) or tetanus toxoid (TT) isolated from MS patients and normal controls. After stimulation with specific antigen but not mitogen, TCLs from HLA-DR2+ donors produced significantly more LT and TNF-alpha than TCLs from DR2- donors. In contrast, HLA-DR2+ and DR2- TCLs did not differ in the production of IFN-gamma, a cytokine also produced by T cells but not encoded in the HLA region. Increased secretion of LT and TNF-alpha was unrelated to the specificity (MBP vs TT), MHC restriction (HLA-DR2 vs other DR molecules), or source (MS vs normal) of the TCLs. There was no significant association of the cytokine production with individual LT or TNF-alpha alleles, indicating that the increased production of these cytokines may be linked to other polymorphic genes in this region. The results suggest that the association of MS with HLA-DR2 implies a genetically determined propensity of T cells to produce increased amounts of LT and TNF-alpha.

Animals↗
PubMed1995

DNA polymerase delta of Physarum polycephalum.

DNA polymerase delta from the phylogenetically ancient slime mold Physarum polycephalum has been 380-fold enriched from amoebae. It was found to have the properties typical for this type of DNA polymerase from higher eukaryotes with regard to effectors, template-primer acceptance, co-purification with 3'-5'-exonuclease activity, as well as the effect of endogenous proliferating cell nuclear antigen (PCNA) from amoebae on the stimulation and processivity of DNA synthesis. An identified cDNA fragment shows 65.5% identical amino acides with DNA polymerase delta from Saccharomyces pombe. The molecular mass of the polymerase is 125 kDa while that of PCNA is 35 kDa. During size-exclusion chromatography, the highly purified polymerase eluted in the position of 125 kDa, suggesting that no other proteins were tightly complexed with the enzyme. The DNA polymerases from the (mononucleate) amoebae and from the (multinucleate) plasmodia of P. polycephalum have very similar properties in contrast to their differences in phenotype and their mode of nuclear division. The polymerase shows a higher degree of similarity than DNA polymerase alpha, and especially the beta-like DNA polymerase, with the corresponding polymerases of higher eukaryotes. According to antibody staining, DNA polymerase delta is readily fragmented by proteases, even in the presence of inhibitor cocktails. Including freshly prepared cell lysates, proteolytic fragments are reproducible, the most abundant being 50 kDa in size. The DNA polymerase is recognized by the antisera against two peptides which have been derived by PCR-screening of plasmodial cDNA. One of the proteolytic splitting sites is located within an eight amino-acid stretch between the two antigenic sequences.

Amino Acid Sequence↗
PubMed1995

Levels of the terminal complement complex, C3a-desArg and C1-inhibitor in adult patients with capillary leak syndrome following bone marrow transplantation.

Capillary leak syndrome (CLS) is a severe complication after bone marrow transplantation (BMT). To investigate whether there is a pathogenetic role of the complement system, we monitored the levels of the terminal complement complex C5b-9 (TCC) and C3a-desArg as indicators of an activation of the complement system and the inhibitor of the classical pathway of the complement cascade, C1 inhibitor (C1-INH), in 48 bone marrow transplant recipients from 1 week before to 5 weeks after transplantation. Capillary leak syndrome developed in 7 out of 48 patients between days 1 and 12 after BMT. Complement activation as indicated by TCC levels was more pronounced in patients with CLS (n = 7) from day -8 to +28 (p < 0.05; day -1) and the elevation of TCC levels lasted longer in CLS patients (peak day 21) than in patients without this complication (peak day 7). Mean C3a-desArg levels were highest in patients with CLS reaching a peak at day 7. During the early posttransplant period a significant elevation of C1-INH levels (p < 0.01 and p < 0.05 respectively) compared with baseline levels (day -8) was found in patients with and without CLS, which was more pronounced in those patients with CLS (p < 0.05). Although we could not observe an absolute C1-INH deficiency as compared to healthy individuals our data support the presence of a relative deficiency of the inhibitor which might explain the reported beneficial effects of C1-INH substitution in BMT related CLS.

Adult↗
PubMed1994

[Hypercoagulability in patients with veno-occlusive disease after bone marrow transplantation].

BACKGROUND: Veno-occlusive disease (VOD) leads to obliteration of small intrahepatic venules and is one of three most important complications with fatal outcome after bone marrow transplantation (BMT). The etiology of VOD is not completely understood. Endothelial cell injury induced by the conditioning myeloablative radiochemotherapy with subsequent activation of the coagulation cascade seems to be a crucial step in the pathogenesis of the disease. PATIENTS AND METHODS: We investigated tissue plasminogen activator (tPA), its main inhibitor (PAI-1) and the natural anticoagulants protein C and S by enzymimmunoassay prospectively in 32 bone marrow transplant recipients. RESULTS: VOD developed in four patients. They presented with extremely elevated levels of PAI-1 after BMT whereas tPA levels remained low. Additionally a transient decrease of protein S was found one week after BMT which was more pronounced in VOD patients. No protein C deficiency was observed. CONCLUSION: Our data suggest that hypofibrinolysis due to an excess of PAI-1 may be involved in the pathogenesis of VOD. The determination of PAI-1 may be useful to recognize the development of VOD and facilitate the decision for thrombolytic therapy with rtPA. A decrease of protein S may play a role as a cofactor in the early phase after BMT.

Bone Marrow Transplantation↗
PubMed1994

A rapid luciferase transfection assay for transcription activation effects and stability control of estrogenic drugs in cell cultures.

A rapid assay system for measuring the potential of estrogenic drugs is introduced. Luciferase induction could be measured in estrogen-receptor-positive human MCF-7 breast cancer cells, which had been transfected with a novel luciferase reporter plasmid ERE luc. The minimal requirement was 1 h exposure to the inducing drug and 3.5 h of incubation after removal of the drug. The assay system was used to measure the stability of the drug diaqua-[1,2-bis (2,6-dichloro-4-hydroxyphenyl) ethylenediamine] platinum(II) sulfate, containing an estrogenic ligand and reactive platinum. Luciferase activity was observed only when the drug was in the culture medium and cells for short times, whereas the estrogenic ligand alone remained active. It is assumed that binding of the platinum moiety to macromolecular constituents of the culture or cells renders the drug inaccessible for binding to the estrogen receptor.

Antineoplastic Agents↗
PubMed1994

1-Carbamoylalkyl-2-phenylindoles: relationship between side chain structure and estrogen antagonism.

The 2-phenylindole system has proved to be a versatile structure for the design of potent antiestrogens, especially when functional groups have been introduced into the alkyl side chain in position 1. In analogy to steroidal structures such as ICI 164,384 a number of 2-phenylindoles with carbamoylalkyl and aminoalkyl side chains were synthesized. They bind to the calf uterine estrogen receptor with relative binding affinities between 2.1 and 21 (estradiol = 100). The antiestrogenic effect of these compounds was demonstrated by the inhibition of transcriptional activity which was measured in a new luciferase assay with the EREwtc luc as reporter plasmid. The derivative with a methyl-n-propyldodecanamide side chain (4h) antagonized the effect of estradiol (10(-9) M) completely at concentrations of 10(-7) M and higher. As a sensitive model for quantification of estrogenic and antiestrogenic effects in vitro we used HeLa-cells cotransfected both with the reporter plasmid and estrogen receptor expression vectors HEG0 and HE0. In cells transfected with these vectors transcriptional activity was strongly dependent on side chain structure. With mutated receptors we were able to show that this activity was mainly due to TAF-1 whereas TAF-2 remained silent. When we studied the effect of some of the new compounds in vivo using the mouse uterine weight assay, we observed a correlation between transcriptional activity in transfected HeLa cells and estrogenic effects in mice. Two of the 1-carbamoylalkyl-2-phenylindoles (4f, 4h) proved to be "pure" antiestrogens both in vitro and in vivo. In estrogen-sensitive MCF-7 breast cancer cells, they strongly inhibit cellular growth. Some of the IC50-values were close to 10(-8) M.

Animals↗
PubMed1994

Ionizing radiation induces human intercellular adhesion molecule-1 in vitro.

Intercellular adhesion molecule-1 (ICAM-1) plays a central role in various inflammatory reactions and its expression is readily induced by inflammatory stimuli such as cytokines or ultraviolet irradiation. We have investigated the effect of ionizing radiation (IR) on human ICAM-1 expression in human cell lines and skin cultures. ICAM-1 mRNA levels in HL60, HaCaT, and HeLa cells were elevated at 3-6 h after irradiation and increased with doses from 10-40 Gy. The rapid induction of ICAM-1 occurred at the level of transcription, was independent of de novo protein synthesis, and did not involve autocrine stimuli including tumor necrosis factor-alpha and interleukin-1. IR also induced ICAM-1 cell surface expression within 24 h. Immunohistologic analysis of cultured human split skin revealed ICAM-1 upregulation on epidermal keratinocytes and dermal microvascular endothelial cells 24 h after exposure to 6 Gy. In conclusion, we propose ICAM-1 as an important radiation-induced enhancer of immunologic cell adhesion, which contributes to inflammatory reactions after local and total body irradiation.

Antibodies↗
PubMed1994

Granulocyte colony-stimulating factor inhibits the endogenous leukotriene production in tumour patients.

Granulocyte colony-stimulating factor (G-CSF) is virtually devoid of inflammatory side-effects when given to patients in therapeutic doses. This is in contrast to other haemopoietic cytokines such as granulocyte-macrophage colony-stimulating factor (GM-CSF) or interleukin-3 (IL-3) which may promote inflammatory reactions by increasing the number and/or activity of monocytes, eosinophils, mast cells and basophils. Inflammatory reactions to GM-CSF and IL-3 appear to be related to an increased formation of leukotrienes, known as potent mediators of allergy and inflammation. Here we report that, in contrast to GM-CSF or IL-3, G-CSF has the potential to inhibit the leukotriene production in vivo. G-CSF may thus act as an anti-inflammatory agent. The differential effects of G-CSF and other haemopoietins on endogenous leukotrienes may be of major clinical significance.

Granulocyte Colony-Stimulating Factor↗
PubMed1994

Differential role of protein kinase C in cytokine induced lymphocyte-endothelium interaction in vitro.

In the present study we investigated the influence of the PKC-inhibitor GF109203X on cytokine- and endotoxin-induced expression of intercellular adhesion molecule 1 (ICAM-1) and on the adhesion of lymphocytes to cytokine-activated endothelial cells. We found that tumour necrosis factor alpha (TNF-alpha)- and lipopolysaccharide (LPS)-induced ICAM-1 expression on a human endothelium-derived cell line (EA.hy926) were unaffected by the PKC-inhibitor and thus appeared to be independent of PKC activation. In contrast, GF109203X significantly reduced ICAM-1 expression induced by interferon-gamma (IFN-gamma) and interleukin-1 (IL-1). The functional relevance of these findings was evaluated in an adhesion assay using human umbilical vein endothelial cells (HUVEC) and peripheral blood mononuclear cells (PBMC). In fact, the IFN-gamma- and IL-1-induced adhesion of PBMC to cytokine treated HUVEC could be down-regulated by the PKC-inhibitor, whereas TNF alpha- and LPS-mediated adhesion was not affected. Additionally, the IL-1-driven ICAM-1 expression on HUVEC as well as the IL-1 induced adhesion of PBMC to HUVEC was found to be TNF-dependent, as both effects could be inhibited by an anti-TNF-alpha monoclonal antibody (MoAb) (MAK195). Based on these data on differential regulation of cytokine-induced lymphocyte-endothelium interactions our study supports the use of PKC-inhibitors as additive modulators in cytokine related pathophysiological conditions.

Cell Adhesion↗
PubMed1994

Repeated administration of a F(ab')2 fragment of an anti-tumor necrosis factor alpha monoclonal antibody in patients with severe sepsis: effects on the cardiovascular system and cytokine levels.

In an uncontrolled clinical trial the effects of repeated administration of the F(ab')2 fragment of a murine monoclonal anti-tumor necrosis factor alpha (TNF alpha)-antibody (MAK 195F) on cytokine levels and the cardiovascular system were studied in 20 patients with severe sepsis. Patients were treated with a total of 11 single dosages of the anti-TNF alpha-antibody intravenously over 5 days using either 1 mg/kg (n = 10) or 3 mg/kg (n = 10). The anti-TNF alpha-antibody was well tolerated in all patients without signs of toxicity and without development of anti-murine antibodies. As assessed by cytokine levels (TNF alpha, Interleukin-6) and hemodynamics there was no evidence that the higher dosage of the anti-TNF alpha-antibody (3 mg/kg per dose) was more effective than the lower dosage (1 mg/kg per dose). Comparison of our data with recent data from phase I or II trials using a complete murine monoclonal anti-TNF alpha-antibody suggest that the F(ab')2 fragments of the murine monoclonal anti-TNF alpha-antibody may be of similar efficacy. Definitive conclusions, however, with respect to improvement of mortality and improvement of the cardiovascular system, await the results of larger ongoing placebo-controlled trials.

Adult↗
PubMed1994

Changes in skeletal muscle pO2 after administration of anti-TNF alpha-antibody in patients with severe sepsis: comparison to interleukin-6 serum levels, APACHE II, and Elebute scores.

In 20 patients with severe sepsis, skeletal muscle pO2 was continuously measured in order to assess whether a decrease of skeletal muscle pO2 was accompanied by an improvement of sepsis after repeated administration of F(ab')2 fragments of a murine anti-TNF alpha-antibody. Abnormally high skeletal muscle pO2 decreased from 43.5 +/- 10.9 mmHg (day 0) to 36.4 +/- 10.1 mmHg within 24 h after the first administration of anti-TNF alpha-antibody (day 1, p = .006, n = 20) and remained at 34.6 +/- 7.7 mmHg thereafter (mean day 2-7, p = .004). The decrease of skeletal muscle pO2 within 24 h exceeded 5 mmHg (-7 to -19 mmHg) in 11 patients in contrast to nine patients (-4 to +4 mmHg). Only in the patients showing a decrease of skeletal muscle pO2 did sepsis improve as determined by Elebute score, APACHE II score, and interleukin-6 serum levels. The change of skeletal muscle pO2 within 24 h was associated with a change of interleukin-6 serum levels within 24 h (r = .5, n = 20), with a change of Elebute score (r = .7, n = 20) and of APACHE II score (r = .62). These data suggest that a decrease of skeletal muscle pO2 might be an early indicator of improvement of sepsis after administration of anti-TNF alpha-antibodies.

Adult↗
PubMed1994

Parameters of the fibrinolytic system in patients undergoing BMT: elevation of PAI-1 in veno-occlusive disease.

Veno-occlusive disease (VOD) represents one of the more frequent and most severe complications after BMT. The pathophysiology of VOD is poorly understood. To investigate a possible link between endothelial cell damage and VOD, tissue plasminogen activator (tPA) and its inhibitor (PAI-1) were measured in 32 patients as endothelial cell-derived parameters of the fibrinolytic system. A nearly fivefold increase (mean 103.9 ng/ml, range 22.6-582.4 ng/ml, p < 0.05) in PAI-1 levels was found in the four patients who developed VOD compared with patients without this complication (mean 22.2 ng/ml, range 1.4-131.6 ng/ml). No significant difference was found in tPA levels between patient groups with or without VOD or other complications following BMT, indicating a shift of the fibrinolytic balance towards hypofibrinolysis particularly in patients with VOD. We conclude that alterations of the fibrinolytic system occur in patients undergoing BMT. Hypofibrinolysis seems to be at least one factor in the pathogenesis of VOD and the determination of PAI-1 might be helpful for diagnosing the disease. Our data also may explain the reported successful treatment of VOD by recombinant tPA.

Bone Marrow Transplantation↗