Biomedical subjects

N A Horn

Publications and source records attributed to N A Horn.

10 recordsLinked to original sources

PubMed2003

Effect of isoflurane on monocyte adhesion molecule expression in human whole blood.

BACKGROUND: Recruitment of monocytes to inflamed tissue is a crucial step in the acute inflammatory reaction. Adherence of monocytes to endothelial cells followed by transmigration depends on monocyte surface adhesion molecules, inflammatory cytokines and chemoattractant chemokines. In the present study, we determined the effect of isoflurane on monocyte adhesion receptor expression in vitro. METHODS: Citrated whole blood was incubated for 60 min with either 0.5 or 1 MAC isoflurane. In unstimulated blood samples and after stimulation with N-formyl-methionyl-leucyl-phenylalanine (FMLP) monocyte cell-surface expression of the selectins PSGL-1 and L-selectin, and the beta2-integrins CD11a and CD11b were evaluated by flow cytometry. RESULTS: Isoflurane reduced significantly the expression of PSGL-1 on unstimulated monocytes, whereas the remaining selectins and beta2-integrins were not affected. At both concentrations, the FMLP-induced removal of PSGL-1 from the monocyte surface was increased. Furthermore, at 1 MAC isoflurane the FMLP-induced increase in CD11a expression was significantly inhibited. The surface expression of L-selectin and CD11b was not affected following exposure to isoflurane. CONCLUSION: Isoflurane increases the removal of the selectin PSGL-1 from the monocyte surface. Since PSGL-1 is important during the initial step of monocyte adhesion to endothelial P-selectin, the decrease in monocyte surface PSGL-1 may have profound effects on monocyte-endothelial interactions. Furthermore, the effects of isoflurane on monocyte adhesion molecule expression are different from those reported for neutrophils.

Anesthetics, Inhalation↗

PubMed2003

The effects of sevoflurane and desflurane in vitro on platelet-leukocyte adhesion in whole blood.

The interaction between platelets and leukocytes plays an important role in inflammatory and thrombotic processes. We investigated whether the volatile anaesthetics sevoflurane and desflurane alter the formation of platelet-leukocyte aggregates and the expression of P-selectin on platelets. Whole blood was incubated with 1 and 2 minimum alveolar concentration (MAC) sevoflurane or desflurane. Unstimulated and adenosine diphosphate, or thrombin receptor agonist peptide-6-stimulated samples were stained with fluorochrome-conjugated antibodies. The formation of platelet-leukocyte conjugates and the expression of P-selectin on platelets were measured using flow cytometry. Sevoflurane was found to enhance the binding of platelets to lymphocytes, neutrophils and monocytes, it also increased the expression of P-selectin on platelets especially in the stimulated samples. Desflurane decreased the percentage of lymphocyte-platelet, neutrophil-platelet and monocyte-platelet conjugates principally in unstimulated samples. The results show that these two volatile anaesthetics have differing effects on the formation of platelet-leukocyte conjugates in vitro. Sevoflurane also enhanced the expression of P-selectin on platelets.

Anesthetics, Inhalation↗

PubMed2001

Coagulation assessment in healthy pigs undergoing single xenon anaesthesia and combinations with isoflurane and sevoflurane.

BACKGROUND: With the introduction of new anaesthetics into clinical practice possible side effects of these novel anaesthetics have to be evaluated. This study was performed to clarify whether xenon or combinations of xenon with isoflurane or sevoflurane modify blood coagulation. METHODS: The study was performed in 20 healthy pigs which first underwent xenon anaesthesia (65 Vol%) and were then randomly assigned to combinations of xenon and isoflurane or sevoflurane at varying concentrations. During anaesthesia the following parameters were controlled: aPTT, PT, fibrinogen concentrations and thrombelastographic measurements. RESULTS: Xenon monoanaesthesia did not alter significantly any coagulation parameter. When isoflurane was introduced the aPTT showed a significant increase while fibrinogen concentration decreased. The introduction of sevoflurane led also to a decrease in fibrinogen concentration, while the aPTT was unchanged. These decreases in fibrinogen concentration were not accompanied by reduced maximal clot strength or elevated fibrinolysis evaluated by thrombelastography. Although the above-described changes were statistically significant, none of the parameters throughout the experiment exceeded the limits of normal values. CONCLUSION: In our study, xenon monoanaesthesia and combinations of xenon with isoflurane and sevoflurane did not lead to pathologic alterations in the measured coagulation parameters.

Anesthesia, Inhalation↗

PubMed2001

Xenon does not affect human platelet function in vitro.

We sought to determine whether xenon affects platelet glycoprotein expression and platelet-related hemostasis in vitro at a clinically relevant concentration. Human whole blood was stimulated with either adenosine diphosphate or the thrombin receptor agonist peptide (TRAP)-6 after incubation with 65% xenon. Halothane at 2 minimum alveolar anesthetic concentration was used as a positive control. Platelet function and activation were evaluated with two-color flow cytometry. The expression of the platelet glycoproteins GPIIb/IIIa, GPIb, and P selectin were detected with fluorochrome-conjugated monoclonal antibodies. In vitro measurement of platelet-related hemostasis under conditions of high shear stress was performed in citrated whole blood with a platelet function analyzer (PFA-100((R))) by using collagen/epinephrine and collagen/adenosine diphosphate cartridges. Xenon did not affect basal or agonist-induced expression of platelet membrane glycoproteins, activation-dependent conformational changes of the GPIIb/IIIa receptor, expression of P selectin, or PFA closure times. In contrast, halothane reduced TRAP-6-induced activation of the GPIIb/IIIa complex. Furthermore, collagen/epinephrine-induced PFA closure time was significantly prolonged. These results demonstrate that xenon does not affect the unstimulated or agonist-induced platelet glycoprotein expression, activation of GPIIb/IIIa, or platelet-related hemostasis.

Anesthetics, Inhalation↗

PubMed2001

Sevoflurane inhibits unstimulated and agonist-induced platelet antigen expression and platelet function in whole blood in vitro.

BACKGROUND: Previous studies have reported conflicting results about the effect of sevoflurane on platelet aggregation. To clarify this point, we investigated the effects of sevoflurane on platelet antigen expression and function in vitro. METHODS: Human whole blood was incubated for 1 h with 0.5 and 1 minimum alveolar concentration sevoflurane, 21% O(2), and 5% CO(2). A control sample was kept at the same conditions without sevoflurane. After stimulation with adenosine diphosphate or thrombin receptor agonist peptide 6, samples were stained with fluorochrome conjugated antibodies, and the expression of platelet glycoproteins GPIIb/IIIa, GPIb, and P-selectin, as well as activated GPIIb/IIIa, were measured with two-color flow cytometry. In addition, platelet function was assessed by means of thromboelastography and using the platelet function analyzer 100. RESULTS: Already in subanesthetic concentrations, sevoflurane inhibits unstimulated and agonist-induced GPIIb/IIIa surface expression and activated GPIIb/IIIa expression on platelets in whole blood. The agonist-induced redistribution of GPIb into the open canalicular system was also impaired by sevoflurane, whereas no effect on P-selectin expression in activated platelets could be found. Sevoflurane significantly reduced the maximum thromboelastographic amplitude. Furthermore, platelet function analyzer 100 closure times were significantly prolonged. CONCLUSION: The results show that sevoflurane significantly impairs platelet antigen expression in vitro. It is especially the inhibition of GPIIb/IIIa expression and activation that impairs bleeding time as reflected in thromboelastographic measurements and platelet function analyzer 100 closure times. The exact inhibitory mechanism remains unclear.

Anesthetics, Inhalation↗

PubMed2001

The influence of hypoalbuminemia on maximal flow rates and transmembrane pressure during plasmapheresis--an in vitro study.

BACKGROUND: Plasmapheresis has been used for the treatment of acute liver failure (ALF). In these patients, hypoalbuminemia is often observed. Since albumin improves the disaggregability of erythrocytes, hypoalbuminemia might deteriorate rheology and thus influence the overall performance of plasmapheresis. METHODS: Hypoalbuminemia was mimicked by using porcine blood because of its physiologically low albumin/globulin ratio (AGR). Filters (n = 16) were integrated in a closed extracorporeal in vitro system. In the control group (n = 8), native porcine blood (AGR 0.8) was used. In the study group (n = 8), we used porcine blood supplemented with human albumin to obtain the human AGR value of 1.2. Two different heparinization protocols were compared in each group (2.5 IU/ml: n = 4 with albumin and n = 4 without albumin versus 5 IU/ml: n = 4 with and n = 4 without albumin). RESULTS: In both heparinization protocols the higher AGR led to lower transmembrane pressure (TMP) levels compared to the lower AGR. The reduced TMPs enabled higher blood flow and filtration rates. CONCLUSION: Maintenance of a physiological AGR in ALF patients might improve the performance of plasmapheresis and--as opposed to raised heparinization--contribute to a safer application.

Animals↗

PubMed2000

Comparative analysis of metabolism of medium- and plasma perfused primary pig hepatocytes cultured around a 3-D membrane network.

Culture media are frequently used in the evaluation of metabolical functions of hepatocytes in hybrid liver support systems (hLSS). However, media compositions differ substantially from those of plasma. Therefore, our study was designed to investigate whether current in vitro studies with medium are suitable to assess the metabolical competence of hLSS-cultures during clinical application as well as to explore whether the cell nutrition with medium provides a suitable modus operandi for stand by cultivation. Paired bioreactor cultures were perfused with either Williams' Medium E (MPB) or human plasma (PPB). About 6x108 primary pig hepatocytes (>97% viability) were cultured in three laboratory scale bioreactors designed according to Gerlach's bioreactor-concept. Different perfusion protocols were initiated after a standardised period allowing for cell attachment and reorganisation in aggregates. Whereas patterns of enzyme release were similar in both protocols the metabolical behaviour was different between MPB (anabolic state) and PPB (catabolic state). Furthermore, compared to MPB the lidocaine-MEGX-tests for PPB demonstrated lower MEGX-concentrations and a different reaction pattern. We conclude that the nutrition of hepatocytes with medium during the stand by period itself might influence the cell function and subsequently the efficacy of the hLSS-treatment during clinical application.

Animals↗

PubMed1996

High-yield production of pBR322-derived plasmids intended for human gene therapy by employing a temperature-controllable point mutation.

Production of large quantities of highly purified plasmid DNA is essential for gene therapy. A low-copy-number pBR322-derived plasmid (VCL1005) was converted to a high-copy-number plasmid (VCL1005G/A) by incorporating a G-->A mutation that affects initiation of DNA replication from the ColE1 origin of replication. Because the phenotypic effect of this mutation is enhanced at an elevated temperature, a further increase in yield was achieved by changing the growth temperature from 37 degrees C to 42 degrees C at mid-log phase during batch and fed-batch fermentation. The combined effect of the single base-pair change and the elevated growth temperature produced an overall yield of 2.2 grams of plasmid DNA available for recovery from a 10-liter fed-batch fermentation compared to 0.03 grams from a 10-liter batch fermentation, a 70-fold increase in yield. The plasmid DNA isolated from this process contained lower levels of RNA and chromosomal DNA contaminants, simplifying downstream processing.

DNA, Bacterial↗

PubMed1995

High level expression and refolding of mouse interleukin 4 synthesized in Escherichia coli.

Mouse Interleukin 4 is a 20-kDa glycoprotein, synthesized by activated T lymphocytes and mast cells, which regulates the growth and/or differentiation of a broad spectrum of target cells of the immune system, including B and T lymphocytes, macrophages, and hematopoietic progenitor cells. Using an inducible recA promoter and the g10-L ribosome-binding site, recombinant non-glycosylated interleukin 4 (IL-4) was expressed as 17% of total cellular protein in Escherichia coli inclusion bodies, as a reduced, inactive 14.5-kDa polypeptide. The protein was refolded and aggregates dissociated when three disulfide bonds were reformed by slowly decreasing the concentration of guanidine hydrochloride and cysteine. The oxidized monomer was purified to homogeneity by sequential ion-exchange and size exclusion chromatography. When compared with native IL-4, E. coli-derived IL-4 displayed an identical specific activity of 4-7 x 10(7) units/mg. This recombinant IL-4 contained a three-amino-acid NH2-terminal extension, which did not affect its biological activity. Purified biologically active protein consisted of three isoforms as shown by two-dimensional gel electrophoresis, with a pI greater than 9.0. These data suggest that neither glycosylation nor the NH2 terminus of mouse IL-4 play a critical role in contributing to its in vitro biological activity.

Amino Acid Sequence↗

PubMed1995

Cancer gene therapy using plasmid DNA: purification of DNA for human clinical trials.

A production method has been developed for the purification of pharmaceutical-grade plasmid DNA for in vivo gene therapy. This method has been applied to the purification of VCL-1005, which is a eukaryotic plasmid expression vector that codes for the production of the HLA-B7 protein. Purified VCL-1005 is formulated with a cationic lipid and injected directly into established tumors of HLA-B7-negative patients with advanced cancers to heighten the patient's immune response against the cancer. The purification of pharmaceutical-grade plasmid DNA requires the development of highly reproducible and scaleable processing methods that meet regulatory standards similar to those required for the manufacture of recombinant protein pharmaceuticals. Defined pharmaceutical standards of purity, potency, efficacy, and safety are routinely met by the process described in this study. The scaleable purification method described here is a combination of highly reproducible unit operations; alkaline lysis, precipitation, and size-exclusion chromatography. The advantages over existing DNA purification methods include improved plasmid purity and the elimination of undesirable process additives such as toxic organic extractants and animal-derived enzymes. The overall process yield of purified plasmid DNA from fermentation through final column purified product is greater than 50%. Contaminating Escherichia coli DNA levels are reproducibly below 1% as measured by Southern analysis. Endotoxin levels are less than 0.03 endotoxin units/micrograms plasmid DNA and residual protein is undetectable. This process was used to produce 100 mg of VCL-1005 for use in an active clinical protocol.

Blotting, Southern↗