Search PubMed⌕ Search

Biomedical subjects

M Thurnher

Publications and source records attributed to M Thurnher.

At least 55 records · Page 3Linked to original sources
PubMed1994

Carbohydrate receptor-mediated gene transfer to human T leukaemic cells.

The mucin-type carbohydrate Tn cryptantigen (GalNAc alpha 1-O-Ser/Thr, where GalNAc is N-acetyl-D-galactosamine) is expressed in many carcinomas, in haemopoietic disorders including the Tn syndrome, and on human immunodeficiency virus (HIV) coat glycoproteins, but is not expressed on normal, differentiated cells because of the expression of a Tn-processing galactosyltransferase. Using Jurkat T leukaemic cells which express high levels of Tn antigen due to deficient Tn galactosylation, we have established the Tn antigen-mediated gene transfer and demonstrate the considerable efficiency of this approach. We used poly(L-lysine) conjugates of the monoclonal antibody 1E3 directed against the Tn antigen to deliver the luciferase and beta-galactosidase reporter genes to Jurkat cells by receptor-mediated endocytosis. Addition of unconjugated 1E3 reduced transfection efficiency in a concentration-dependent manner and incubation with free GalNAc abolished DNA transfer completely, indicating that gene delivery is indeed mediated by the Tn antigen. Pre-treatment of Jurkat cells with Vibrio cholerae sialidase, which uncovers additional Tn antigens, resulted in an improvement of gene transfection. Both human and chicken adenovirus particles attached to the DNA/polylysine complex strongly augmented transgene expression. When the beta-galactosidase (lacZ) gene was delivered to Jurkat cells by Tn-mediated endocytosis, up to 60% of the cells were positive in the cytochemical stain using 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal) as a chromogenic substrate. The efficiency of the transferrin receptor-mediated DNA uptake into Jurkat cells was comparatively low, although these cells were shown to express considerable amounts of transferrin receptor. We show here that a mucin-type carbohydrate antigen mediates highly efficient DNA uptake by endocytosis into Jurkat T cells. This method represents a 50-fold improvement of Jurkat cell transfection efficiency over other physical gene transfer techniques. Specific gene delivery to primary cancer cells exhibiting Tn epitopes may especially be desirable in immunotherapy protocols.

Adenoviridae↗
PubMed1994

Pulmonary reaction during intramedullary fracture management in traumatic shock: an experimental study.

Immediate nailing of shaft fractures in severely injured patients causes fat embolization. This method therefore is considered potentially dangerous, since fat intravasation in association with multiple trauma and subsequent endotoxemia might lead to pulmonary dysfunction. We therefore studied the pathophysiologic events of intramedullary nailing in the lungs of sheep with chronic instrumentation including lung lymph fistula. In the 7 animals in group I closed nailing of the intact tibia and femur was performed. Group II (n = 7) animals sustained hypovolemic shock and retransfusion prior to nailing, while group III (n = 11) animals were treated like those in group II and further challenged on the following two days with endotoxin. Group III was compared with group IV (n = 6), in which endotoxin was given only once without additional trauma. Nailing in group I led to a significant increase of the MPAP from 10.8 to 13.8 mm Hg postoperatively (p < 0.05), but no increase in lung permeability. Only additional hypovolemia, retransfusion and nailing as performed in groups II and III showed significant increase of the lymph flow (QI) from 4.4 mL/h to 12.4 mL/h and the protein clearance (Pclear) from 3 to 6.3. A significant difference of the pulmonary permeability between group I and II was only observed postoperatively. There was no difference in the lung response between group III and IV. This ovine study corroborates that although nailing causes a moderate increase in pulmonary pressure, it does not lead to increased lung permeability. Only additional hemorrhagic shock, even when adequately resuscitated, leads to lung disturbance postoperatively. The subsequent endotoxin challenge does not aggravate lung injury.

Animals↗
PubMed1994

Differences in the regulation of specific glycosylation in the pathogenesis of paroxysmal nocturnal hemoglobinuria and the Tn-syndrome.

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hematologic disorder that resembles in several aspects the Tn-syndrome, in which bone marrow-derived cells are deficient in mucin-type beta 1,3 galactosyltransferase (beta 1,3Gal-T) due to the persistent repression of an intact allele. In the present study, we have investigated phytohemagglutinin (PHA)-activated T cells from the peripheral blood of an individual with the well-established clinical diagnosis of PNH. Only 10% of T cells were deficient in surface expression of the glycosylphosphatidylinositol (GPI)-linked CD48 antigen; in contrast, 95% of the patient's polymorphonuclear leukocytes harbored the defect. The cell-surface density of CD48 on unaffected T cells from this patient was two- to three-fold higher when compared to PHA-activated normal donor T cells. CD48-negative T cells were cloned and shown to belong to the CD4+ or the CD8+ antigenic subset. To test for the possibility that in PNH, as in Tn syndrome, gene repression may be responsible for the deficiency, CD48- T cell clones were treated with 5-azacytidine (5-azaC) and sodium n-butyrate (NaB). While such treatment reproducibly led to reexpression of a sialylated CD43 epitope on affected Tn-syndrome T cell clones, these drugs failed to induce reexpression of CD48 on affected PNH T cell clones. Our data suggest that despite many similarities, different pathogenetic mechanisms are responsible for PNH and Tn-syndrome. These CD48- T cell clones are the first to be described and may be useful to define the PNH lesion at a biochemical and genetic level.

Adult↗
PubMed1994

[Pathophysiologic consequences of various osteosynthesis procedures in polytrauma patients. Part I: Experimental studies of intramedullary pressure development in reamed and unreamed intramedullary nailing and plate osteosynthesis of the femur].

The aim of this study was to measure the intramedullary pressure (IP) of a) a reamed intramedullary nail (RN), b) an unreamed intramedullary nail (UN), and c) the AO-plate (PL) for stabilization of an experimental fracture of a sheep femur. The increase in IP whilst reaming or introducing intramedullary nails in long bone fractures is often said to be responsible for fat intravasation and secondary systemic and pulmonary damage after severe blunt trauma. 21 sheep were studied, 7 in each group (RN, UN, PL). The animal study was permitted by the local authorities. Under general anesthesia, the femoral fractures were produced by an oscillating saw, osteosynthesis was then performed whilst continuously measuring IP with a supracondylar IP-transducer-device. The results show similar intramedullary pressures measured during RN and during UN (maximal IP: UN: 203 mmHg, range 76-330; RN: 212 mmHg, range 70-425). The alternative extramedullary PL is not accompanied with any relevant increase of IP (maximal IP: 35 mmHg, range 16-70). In conclusion, RN and UN are not safe procedures concerning the danger of fat-intravasation by IP.

Animals↗
PubMed1993

Use of O-glycosylation-defective human lymphoid cell lines and flow cytometry to delineate the specificity of Moluccella laevis lectin and monoclonal antibody 5F4 for the Tn antigen (GalNAc alpha 1-O-Ser/Thr).

The Tn antigen (GalNAc alpha 1-O-Ser/Thr) is a disease-related O-linked (mucin-type) carbohydrate neoantigen which is expressed in idiopathic Tn syndrome, AIDS, T-cell lymphoma and in many carcinomas. In the present study, we took advantage of a Tn antigen expressing T-lymphocyte clone derived from a patient with the idiopathic form of the Tn syndrome and the Tn+ Jurkat cell line to characterize new reagents that should identify Tn antigens (monoclonal antibody 5F4 and a lectin newly isolated from Moluccella laevis seeds). Flow cytometry revealed that both reagents strongly bound to Tn antigen expressing T lymphocytes but not to normal donor T cells, which are Tn negative. In contrast to mAb 5F4, Moluccella laevis lectin weakly bound to normal donor cells after sialidase pretreatment, indicating its broader specificity. N-Acetyl-D-galactosamine at a concentration of 100 mM significantly reduced antibody binding and abolished lectin binding, completely demonstrating the sugar specificity of both reagents. These reagents should be useful tools in glycobiology and for clinical purposes.

Antibodies, Monoclonal↗
PubMed1993

Persistent repression of a functional allele can be responsible for galactosyltransferase deficiency in Tn syndrome.

A human hematopoietic disorder designated as Tn syndrome or permanent mixed-field polyagglutinability has been ascribed to a stem cell mutation leading to a specific deficiency of UDP-Gal:GalNAc alpha 1-O-Ser/Thr beta 1-3 galactosyltransferase (beta 3 Gal-T) activity in affected cells. To test for the possibility that an allele of the beta 3Gal-T gene might be repressed instead of mutated, we have investigated whether 5-azacytidine or sodium n-butyrate, both inducers of gene expression, would reactivate expression of beta 3Gal-T in cloned enzyme-deficient T cells derived from a patient affected by the Tn syndrome. Flow cytometry revealed that a single treatment induced de novo expression of the Thomsen-Friedenreich antigen (Gal beta 1-3GalNAc-R), the product of beta 3Gal-T activity. In addition, a sialylated epitope on CD43 (leukosialin), which is present on normal but not on beta 3Gal-T-deficient T cells, was also reexpressed. Although no beta 3Gal-T activity was detectable in untreated Tn syndrome T cells, after exposure to 5-azaC,beta 3Gal-T activity reached nearly normal values. Both agents failed to reactivate beta 3Gal-T in Jurkat T leukemic cells, which also lack beta 3Gal-T activity. These data demonstrate that Tn syndrome T cells contain an intact beta 3Gal-T gene copy and that the enzyme deficiency in this patient is due to a persistent and complete but reversible repression of a functional allele. In contrast, the cause of beta 3Gal-T deficiency appears to be different in Jurkat T cells.

Alleles↗
PubMed1992

T cell clones with normal or defective O-galactosylation from a patient with permanent mixed-field polyagglutinability.

To delineate the extent of O-galactosyltransferase deficiency within the lymphoid lineage, monoclonal antibody specific for the Thomsen-Friedenreich (TF) antigen (Gal beta 1----3GalNAc alpha 1-O-Ser/Thr) and its precursor the Tn antigen (GalNAc alpha 1-O-Ser/Thr) were applied to the flow cytometric analysis of peripheral blood lymphocytes from a patient with permanent mixed-field polyagglutinability (PMFP). We show that only a minor population of 4% expressed the Tn antigen which is in contrast to 93% of the patient's erythrocytes carrying the defect. Tn+ lymphocytes mainly belonged to the CD3+ subset, but were also CD19+ or CD16+. Both Tn+ and TF+ T cell clones from patient R. R. were established and shown to belong to the CD4+ or CD8+ antigenic subset. Three glycosyltransferase activities were determined in lysates from these clones: all Tn+ clones were deficient in UDP-Gal: GalNAc alpha 1-O-Ser/Thr beta 1----3 galactosyltransferase (beta 3Gal-T) activity; by contrast this activity was present in all lysates from TF-expressing clones. UDP-GalNAc: polypeptide alpha-N-acetylgalactosaminyltransferase (GalNAc-T) and UDP-Gal: GlcNAc-R beta 1----4 galactosyl-transferase (beta 4Gal-T) exhibited similar activities in both Tn+ and TF+ T cell clones. As a consequence of defective O-galactosylation in Tn+ T cells, cell surface sialic acid of Tn+ clones was reduced by greater than 50% when compared to TF+ clones as demonstrated by sialic acid-specific labeling using fluoresceinated Limax flavus agglutinin(LA) and flow cytometry. The Tn phenotype of T cell clones was stable for more than 1 year of continuous expansion in vitro. These data demonstrate that in PMFP, T cells may also be affected by the O-galactosyltransferase deficiency which is accompanied by a substantial loss of cell surface sialic acid. However, the frequency of Tn+ lymphocytes in peripheral blood from patient R.R. was strikingly low. These T cell clones should be useful to study the defect at a genetic level and the importance of O-linked carbohydrates for proper T cell function.

Agglutination↗
PubMed1991

Decalcified and undecalcified cancellous bone block implants do not heal diaphyseal defects in dogs.

UNLABELLED: In a previous study it has been shown that granulae of decalcified bone matrix do not induce bony healing of 8-week-old mid-diaphyseal defects in dogs. The aim of this study was to test whether osteoinduction combined with the osteoconductive mechanisms provided by the natural structure of cancellous bone blocks would yield better results. A 30-mm-long diaphyseal defect of the left ulna was created in eight adult mongrel dogs and the bone was stabilized with a plate. A Silastic spacer was inserted in the defect for 8 weeks, followed by implantation of frozen undecalcified or decalcified allogeneic cancellous bone blocks for 16 weeks. Healing was analyzed using morphologic methods. At 16 weeks after implantation all grafts had been resorbed. In the decalcified group one defect healed, while none in the other group did so. The implant material was bioassayed in nude rats for osteoinductivity, which was found to be low in decalcified matrix and not detectable in undecalcified bone. CONCLUSION: Allogeneic cancellous bone blocks, demineralized or not, have no osteoinductive capacity and no osteoconductive function that promotes healing of mid-diaphyseal bone defects in dogs.

Animals↗
PubMed1991

Fresh autogeneic, frozen allogeneic, and decalcified allogeneic bone grafts in dogs.

In fully-grown mongrel dogs, diaphyseal ulnar defects 25 mm long were stabilised by screws and plates, and were temporarily filled with silicone rubber blocks. After eight weeks the block was replaced either by fresh autogeneic cancellous bone, allogeneic deep-frozen cancellous bone, allogeneic decalcified bone matrix, or bone matrix gelatin. After 24 weeks the implants were evaluated by radiography, histology, and measurements of new bone volume, using computer-assisted density registration on microradiographs. Only the autogeneic bone grafts led to healing in all instances. Bone regeneration in the other groups was not significantly better than in the sham group in which no graft was employed. Decalcified bone matrix proved ineffective.

Animals↗
PubMed1990

Effect of the PAF antagonist BN 52021 in ovine endotoxin shock.

It has been reported that a platelet-activating factor (PAF) antagonist ONO-6240 had little effect on pulmonary hypertension and lung lymph in sheep injected with endotoxin lipopolysaccharide (LPS); consequently we evaluated a new PAF antagonist, BN 52021. Twelve adult sheep were surgically prepared for chronic study including lung lymph drainage. LPS (S. abortus equii, 36 ng/kg/h over 7 h) was then administered with (BN group) or without (control group) infusion (1 h before LPS) of the PAF antagonist BN 52021 (4 mg/kg/30 min). Comparison of the hemodynamic data between the control group and the BN group reveals an almost identical pattern. In contrast, we find differences in the pulmonary response of the two experimental groups. The early elevation of pulmonary artery pressure after LPS administration is less in the treatment group, as is the duration of pulmonary hypertension. Similarly, thromboxane levels are lower in the treated animals. The increases in lymph flow (LQ) and transvascular protein clearance (L/P X LQ) as well as the protein flux were less prominent in the treatment group. The mean lymph flow per hour was significantly higher in the control group. The mean lymph flow per hour was significantly higher in the control group, as were the corresponding parameters for clearance and protein flux. Thus the pulmonary vasculature and lung fluid balance disruption produced by LPS was markedly reduced by treatment with the PAF blocker.

Animals↗
PubMed1989

The influence of fibrin sealant on demineralized bone matrix-dependent osteoinduction. A quantitative and qualitative study in rats.

Allogeneic demineralized bone matrix (DBM) and bone matrix gelatin (BMG) were implanted with or without fibrin sealant (FS) ectopically (abdominal wall) and orthotopically (7-mm trepanation defect) in 38 male Sprague-Dawley rats. Evaluation was done by descriptive histology, histomorphometry of orthotopic implants, and determination of alkaline phosphatase in ectopic implants. The observation period was 21 days with ectopic implantation and 26 days with orthotopic implantation. In all ectopic specimens, new bone developed without any qualitative difference between specimens with and without FS. The alkaline phosphatase activity did not change significantly upon addition of FS. Morphometry revealed slight differences between the groups with and without FS. The peripheral bone deposits in the BMG + FS group, was significantly larger than in the BMG group. These investigations demonstrated neither a clearly positive nor negative effect of FS on ectopic osteoinduction or BMG-dependent osteoregeneration.

Alkaline Phosphatase↗
PubMed1989

Cardiovascular reaction pattern during endotoxin or peptidoglycan application in awake sheep.

Peptidoglycans (PG) and lipopolysaccharides (LPS) are cell wall constituents of bacteria. The relative contents vary significantly between gram-negative and gram-positive strains. Since both strains take part in clinical septicemia, we compared hemodynamic and permeability properties in a sheep model with chronic instrumentation and lung lymph drainage. The cardiovascular reaction patterns of PG and LPS were comparable at very different dose levels, with LPS being 10,000-fold more potent. Continuous infusion produced an increase in lung permeability (only with LPS) and cardiac output, as well as fever, to mimic the hyperdynamic situation in early septicemia.

Animals↗
PubMed1988

The primary structure of human ribonuclease/angiogenin inhibitor (RAI) discloses a novel highly diversified protein superfamily with a common repetitive module.

Immunological screening of a lambda gt11 library, constructed from HeLa mRNA, yielded several ribonuclease/angiogenin inhibitor (RAI) cDNA clones containing 900-bp inserts. Northern blot analysis revealed that the length of the RAI mRNA is approximately 1.9 kb. Construction and screening of a eukaryotic cDNA expression library (HeLa) containing preferentially complete cDNA inserts led to the isolation of a full length clone. The complete nucleotide sequence was determined. The C-terminal amino acid sequence deduced from the cDNA is identical to the peptide sequence obtained from a CNBr fragment of RAI, confirming the identity of the clone. The deduced primary structure of RAI consists of eight homologous tandem repeats with remarkable periodicity of leucine and cysteine residues. Each repeat is derived from the duplication of a leucine-rich 28-amino-acid module. This prototype module is closely related to a repetitive 24-amino-acid motif of unclear function, previously found in proteins involved in important biological processes such as blood coagulation, embryonic development, cell morphogenesis and signal transduction. Although homologous, the RAI modules show distinct differences in length and amino acid composition to the modules of this group of proteins, demonstrating their high potential of variability, necessary for adaptation to very diverse roles. Based on our results we propose that these repetitive modules are a common structural feature of a novel protein superfamily whose members exert their function by highly specific protein-protein interactions.

Amino Acid Sequence↗
PubMed1988

Control of perfusion pressure and flow in isolated heart bioassays.

Bioassays using isolated animal hearts are important tools for the investigation of cardiac behaviour, but to obtain accurate results a proper perfusion circuit has to be designed. In particular, biophysical studies of contractile and vascular behaviour require a perfusion circuit which permits the adjustment of several experimental parameters within wide ranges. It must also be able to maintain the stability of these parameters when the behaviour of the isolated organ undergoes major changes. To meet this requirement, we have developed a perfusion circuit which makes it possible to control either the perfusion pressure or the coronary flow, with a high degree of precision. There is an electronic controller which satisfies the requirements of a variety of safety and experimental requirements and guarantees a well-defined perfusion system. Computer simulation of the interaction between the perfusion circuit and the heart identified the basic elements of this time-variable, nonlinear system.

Animals↗