Biomedical subjects

M Brandl

Publications and source records attributed to M Brandl.

At least 19 recordsLinked to original sources

PubMed2006

New methods for hemoglobin detection in a microparticle-plasma suspension.

In the extracorporeal adsorption system, MDS (Microspheres based Detoxification System), micro-adsorbent particles measuring 1-25 micrometers circulate in a filtrate circuit for highly specific blood purification/adsorption. The MDS circuit containing the adsorbent microparticles is linked to the patient's blood line by a hollow fiber plasma filter. When the transmembrane pressure or the shear forces due to the red blood cells in the hollow fiber filter are too high, they can be damaged and hemoglobin will be released. In order to detect free hemoglobin (fHb) by optical means, we have designed a new flow-dynamic filter system, placed in the microadsorbent circuit for continuous separation of microparticles from the filtrate. In the flow dynamic filter, we use a high velocity liquid vortex to remove sedimentation and particle plugs on the filter membrane. In our investigations, 3 and 8 micron cellulose nitrate filter membranes for particle separation are used. The obtained particle free bypass filtrate flow rates are typically 0.5 and 0.8 ml/min respectively. The typical sensitivity for fHb detection by the applied noninvasive optical method is 0.15 g/dL. Medical safety regulations require a fail-safe mechanism for fHb detection which monitors the bypass filtrate flow in the flowdynamic filter and shuts down the system in case of membrane occlusion. The bypass filtrate flow is monitored by periodically occluding and releasing the bypass line by means of a clamp. The resulting back pressure profile gives information about the actual filtration rate. This safety principle was proven by statistical analysis and shows its clear functionality.

Adsorption↗

PubMed2005

Detection of fluorescently labeled microparticles in blood.

BACKGROUND: A microsphere-based detoxification system is an adsorption system, whereby microadsorbent particles having diameters of 1-20 microm circulate in an extracorporeal filtrate circle. A thin-wall hollow-fiber membrane filter separates the microparticle-plasma suspension from the bloodstream. For patient safety, it is necessary to have a means to detect membrane ruptures that could lead to a release of microparticles into the patient's bloodstream. METHODS: An optical detection system was developed to monitor the venous bloodstream for the presence of microparticles from the filtrate circuit. For detection purposes, cellulose microspheres, both ferromagnetic and fluorescence labeled, were included with the microsphere adsorbant particles. In the case of a membrane rupture, the labeled particles would also be released into the bloodstream. By illuminating a small volume of blood with an excitation wavelength (590 nm) of the fluorescence marker, the particles can be detected by their emission light at 620 nm. The detector sensitivity is increased by collecting the ferromagnetic and fluorescently labeled microparticles using a magnetic trap. The efficiency of magnetic trap arrangements was tested by adjusting the magnet placements. RESULTS: In vitro experiments were performed by pumping whole blood and labeled microparticles through the fluorescence detector. The efficiency of a magnetic trap arrangement was determined. With an optimal trap setup, 5-10 microl of labeled microparticles can be clearly detected in streaming whole blood. CONCLUSION: An easy to handle microparticle detector was developed, ready for use in particle based blood detoxification systems. The microparticle detection system fulfills the medical and technical requirements to bring the MDS into clinical tests.

Blood Chemical Analysis↗

PubMed2005

Porcine gastric mucin binds to recombinant norovirus particles and competitively inhibits their binding to histo-blood group antigens and Caco-2 cells.

AIMS: To determine if human noroviruses (NV) bind to histo-blood group antigens (HBGA) from pig gastric mucosa. METHODS AND RESULTS: An assay was developed to measure the inhibition of binding of recombinant norovirus-like particles (rNVLP) to HBGA in human saliva by porcine gastric mucin (PGM). The binding of rNVLP to HBGA could be inhibited by PGM in a dose-dependent pattern. Also, rNVLP could be captured effectively by PGM coated directly on plates and was detected by binding of polyclonal antibodies against rNVLP. Similarly, the binding of rNVLP to PGM could be inhibited effectively by HBGA in human saliva, and by Lewis b and Lewis d synthetic oligosaccharides (OS), but not inhibited effectively by an H3 OS or by purified bovine submaxillary gland mucin. Preincubation of rNVLPs with PGM completely inhibited their binding to human Caco-2 cells. CONCLUSIONS: PGM binds effectively to rNVLPs and competitively inhibits rNVLPs binding to human HBGA and Caco-2 cells. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of the binding of glycoproteins from animal gastric mucosa to human NVs. This study highlights the importance of further characterizing the NV incidence and infections in nonhuman animal hosts and the possibility that NV is a zoonotic disease.

Animals↗

PubMed2003

5-Fluorouracil in vesicular phospholipid gels for anticancer treatment: entrapment and release properties.

Vesicular phospholipid gels (VPG), i.e. highly concentrated liposomal dispersions, are suitable for entrapping substances such as anticancer drugs with particular high encapsulation efficiencies (EE). We prepared different formulations of VPG with 30% (w/w) lipid containing 5-fluorouracil (5-FU) by high pressure homogenization and analysed their EE and drug release. Using mixtures of hydrogenated soy phosphatidylcholine and cholesterol with molar ratios ranging from 55/45 to 75/25, a decreasing amount of cholesterol correlated with an increasing EE, which is probably due to a reduced amount of smaller vesicles and number of lamellae. Using a 5-FU solution of pH 8.6 for VPG preparation, an EE of approximately 40% was found after redispersion of the gel to a liposomal dispersion and separation of free drug from liposomal drug by size exclusion chromatography. The reduced EE for preparations with lower pH values was attributed to a fast initial drug release due to the increased drug lipophilicity below the pK(a) value of 8. After redispersion of a VPG of pH 8.0, an initially faster release of about a third of the entrapped drug was found during the first 20 min, followed by stable entrapment over many hours. The rapid initial release may be due to the portion of liposomes smaller than 40 nm in diameter, determined by photon correlation spectroscopy. Cryo electron microscopic pictures show a lentil-like shape of these small liposomes. The membrane defects on the edges are probably the reason for the very high initial drug release rate. The half-life time of the release of 5-FU from intact FU-VPG at both pH 7.4 and 8.0 was found to be in the order of 4-5 h and the kinetics are typical for matrix-controlled drug diffusion. The in vitro data of 5-FU loaded VPG suggest their applicability as implants with controlled release properties or, after redispersion, as intravenously injected liposomal formulations.

Antimetabolites, Antineoplastic↗

PubMed2002

Change in pharmacokinetic and pharmacodynamic behavior of gemcitabine in human tumor xenografts upon entrapment in vesicular phospholipid gels.

PURPOSE: The in vivo pharmacokinetics (PK), biodistribution and antitumor activity of a new liposomal formulation of gemcitabine (GemLip) were compared to the conventional (clinical) formulation of gemcitabine (GemConv). METHODS: Gemcitabine was entrapped in a vesicular phospholipid gel (VPG) consisting of densely packed liposomes. Redispersed VPG containing GemLip consisted of 33% liposomally entrapped and 67% free gemcitabine. The in vivo efficacies of GemLip and GemConv were compared using the subcutaneously growing human soft tissue sarcoma SXF 1301 and the orthotopically growing human bladder cancer BXF 1299T. PK and biodistribution were evaluated using radiolabeled drug and lipid in SXF 1301 tumor-bearing nude mice. RESULTS: GemLip was highly active in SXF 1301 at a gemcitabine dose of 6-9 mg/kg (days 1, 8 and 15; dose near the MTD). In the 6-mg/kg groups, complete tumor remissions were observed in seven of eight mice. Equimolar doses of GemConv resulted in only moderate tumor growth inhibition. Even at equitoxic doses (360 mg/kg given on days 1, 8 and 15, or 120 mg/kg on days 1, 5 and 8) GemConv was less active than GemLip. Furthermore, GemLip was active in the orthotopically growing BXF 1299T bladder cancer model at 6 mg/kg and prevented distant organ metastasis. In the PK study, GemLip achieved a 35-fold higher plasma AUC (1680 mg x h/ml) than GemConv (47.6 mg x h/ml). The serum half-lives were 0.15 h for free gemcitabine and 13.3 h for liposomal gemcitabine (6 mg/kg each i.v.). Moreover, gemcitabine levels in tumors were fourfold higher following injection of GemLip than following injection of GemConv. CONCLUSIONS: GemLip is a highly effective gemcitabine delivery system which results in superior gemcitabine pharmacodynamics and PK than GemConv. The enhanced in vivo efficacy might be explained by sustained release and passive tumor targeting.

Animals↗

PubMed2001

Quantitative imaging of tumour blood flow by contrast-enhanced magnetic resonance imaging.

Tumour blood flow plays a key role in tumour growth, formation of metastasis, and detection and treatment of malignant tumours. Recent investigations provided increasing evidence that quantitative analysis of tumour blood flow is an indispensable prerequisite for developing novel treatment strategies and individualizing cancer therapy. Currently, however, methods for noninvasive, quantitative and high spatial resolution imaging of tumour blood flow are rare. We apply here a novel approach combining a recently established ultrafast MRI technique, that is T(1)-relaxation time mapping, with a tracer kinetic model. For validation of this approach, we compared the results obtained in vivo with data provided by iodoantipyrine autoradiography as a reference technique for the measurement of tumour blood flow at a high resolution in an experimental tumour model. The MRI protocol allowed quantitative mapping of tumour blood flow at spatial resolution of 250 x 250 microm(2). Correlation of data from the MRI method with the iodantipyrine autoradiography revealed Spearman's correlation coefficients of Rs = 0.851 (r = 0.775, P < 0.0001) and Rs = 0.821 (r = 0.72, P = 0.014) for local and global tumour blood flow, respectively. The presented approach enables noninvasive, repeated and quantitative assessment of microvascular perfusion at high spatial resolution encompassing the entire tumour. Knowledge about the specific vascular microenvironment of tumours will form the basis for selective antivascular cancer treatment in the future.

Animals↗

PubMed2001

Filter extrusion of liposomes using different devices: comparison of liposome size, encapsulation efficiency, and process characteristics.

Liposomes were prepared by stepwise extrusion through 5, 1, 0.4, 0.2, 0.1 and 0.05 microm pore sizes using two different filter-extruders, the continuous high pressure device Dispex Maximator (CE) or alternatively the discontinuous Avestin LiposoFast (DE). The liposome dispersions obtained were compared in terms of particle size, lamellarity and encapsulation efficiency of calcein. The liposomes were smaller with CE than DE at all stages due to higher flow rates and pressure drops, except for final filter pore size (0.05 microm) where both preparations had similar sizes. The particle size analysis technique itself had a strong influence on the liposome sizes measured. For bigger liposomes (extruded through 0.4 microm filters) the Nicomp 370 revealed bigger volume-based mean particle sizes along with more stringent differences between volume-based and number-based diameters than the Malvern Zetasizer. In contrast, for small liposomes extruded through 0.05 microm filters, similar liposome sizes were found no matter which of the two PCS techniques or cryo-transmission electron microscopy was used. In congruence to the liposome sizes measured, encapsulation efficiencies were smaller for CE than DE at all filter stages except the final (0.05 microm). No lipid loss occurred and lyso-phosphatidylcholine formation was negligible irrespective of which extrusion technique was used.

Filtration↗

PubMed2001

Steam sterilisation of vesicular phospholipid gels.

Vesicular phospholipid gels (VPGs), highly concentrated phospholipid dispersions of semisolid consistency and vesicular morphology are under investigation as potential implantable depots for sustained release of drugs and as intermediates for subsequent dilution into 'conventional' liposome dispersions. It was investigated here if VPGs can be steam sterilised. VPGs prepared from 400 mg/g egg-phosphatidylcholine by high-pressure homogenisation retained their vesicular structure but showed a slight increase in vesicle size (freeze-fracture electron microscopy). However, autoclaving slowed down both, the in vitro release of the hydrophilic marker carboxyfluorescein and vesicles from VPGs. This was assumed to be due to bigger vesicle sizes and corresponding increase in packing density of the vesicular matrix. Upon dilution into a liposome dispersion both negative staining electron microscopy and dynamic laser light scattering analysis confirmed a distinct increase in liposome size, mainly due to fusion of small (20 nm) vesicles with unfavourable curvature. This was consistent with the observed increase in encapsulation efficiency of carboxyfluorescein. Phospholipid hydrolysis during autoclaving was negligible with lysophosphatidylcholine formation of less than 2% (thin layer chromatography). Despite significant change of their morphological and functional properties during autoclaving VPGs retained their main characteristics, such as vesicular structure, sustained release and dilutability to liposome dispersions, and are, therefore, considered as autoclavable.

Fluoresceins↗

PubMed2001

C-H...pi-interactions in proteins.

A non-redundant set of 1154 protein structures from the Protein Data Bank was examined with respect to close interactions between C-H-donor and pi-acceptor groups. A total of 31,087 interactions were found to satisfy our selection criteria. Their geometric parameters suggest that these interactions can be classified as weak hydrogen bonds.A set of 12 interaction classes were defined based on the division of the donors into three groups and the acceptors into four groups. These classes were examined separately, and the respective interactions described in detail in each class. Most prominent were interactions between aliphatic C-H donors and aromatic pi-acceptors and interactions between aromatic C-H donors and aromatic pi-acceptors. About three-quarters of the Trp-rings, half of all Phe and Tyr-rings and a quarter of all His-rings were found to be involved as acceptors in C-H...pi-interactions. On the donor side, a preference for aromatic C-H groups was observed, but also for the aliphatic side-chains of the long, extended amino acid residues Lys, Arg and Met, and the Pro ring. The average distance between the C-donor and the center-of-mass of the pi-acceptor was observed to be significantly longer in the 174 protein structures determined at >2.5 A resolution. Also, the distribution is significantly wider. This resolution dependence suggests that the force fields commonly used for the refinement of protein structures may not be adequate. C-H...pi-interactions involving aromatic groups either as donor or as acceptor groups are found mostly in the interior of the protein. The more hydrophilic the participating groups are, the closer to the surface are the interactions located. About 40 % of all C-H...pi-interactions occur between amino acid residue side-chains that are separated by nine or less residues in sequence. Dependent on the interaction class, different preferences for secondary structure, residue type and side-chain conformation were observed. It is likely that the C-H...pi-interactions contribute significantly to the overall stability of a protein.

Databases, Factual↗

PubMed2001

An outbreak of Salmonella serotype Thompson associated with fresh cilantro.

An outbreak of Salmonella serotype Thompson in California was identified through laboratory-based surveillance and investigated with case-control, traceback, and laboratory studies. There were 35 "sporadic" cases and a restaurant-associated outbreak of 41 cases with onset between 6 March and 31 March 1999. Three case patients were hospitalized. A case-control study found a significant association between illness and eating cilantro at a restaurant (63% of case patients vs. 34% of control subjects; odds ratio, 3.5; 95% confidence interval, 1.1-11.4). Although common distributors of cilantro were identified, inadequate records prohibited the identification of a single farm supplying cilantro. At room temperature, Salmonella Thompson grew more rapidly and to a higher concentration on chopped cilantro, compared with whole-leaf cilantro. Freshly made salsa (pH 3.4) supported growth of Salmonella Thompson. Cilantro should be served promptly after chopping. Accurate records of the distribution of produce should be available, and bacterial contamination of produce should be prevented in retail and wholesale establishments, in packing sheds, and on farms.

Adolescent↗

PubMed2001

Molecular dynamics simulation reveals conformational switching of water-mediated uracil-cytosine base-pairs in an RNA duplex.

A 4 ns molecular dynamics simulation of an RNA duplex (r-GGACUUCGGUCC)(2 )in solution with Na+ and Cl- as counterions was performed. The X-ray structure of this duplex includes two water-mediated uracil-cytosine pairs. In contrast to the other base-pairs in the duplex the water-mediated pairs switch between different conformations. One conformation corresponds to the geometry of the water-mediated UC pairs in the duplex X-ray structure with water acting both as hydrogen-bond donor and acceptor. Another conformation is close to that of a water-mediated UC base-pair found in the X-ray structure of the 23 S rRNA sarcin/ricin domain. In this case the oxygen of the water molecule is linked to two-base donor sites. For a very short time also a direct UC base-pair and a further conformation that is similar to the one found in the RNA duplex structure but exhibits an increased H3(U)...N3(C) distance is observed. Water molecules with unusually long residence times are involved in the water-mediated conformations. These results indicate that the dynamic behaviour of the water-mediated UC base-pairs differs from that of the duplex Watson-Crick and non-canonical guanine-uracil pairs with two or three direct hydrogen bonds. The conformational variability and increased flexibility has to be taken into account when considering these base-pairs as RNA building blocks and as recognition motifs.

Base Pairing↗

PubMed2001

Local immunotherapy of glioma patients with a combination of 2 bispecific antibody fragments and resting autologous lymphocytes: evidence for in situ t-cell activation and therapeutic efficacy.

After adoptive transfer of pre-activated lymphocytes into the operation cavity of glioma patients, tumor regression and improved survival have been reported in some patients. Results were most impressive when bispecific antibodies with tumor x CD3 specificity were also applied. In this study, we attempted to avoid time-consuming pre-activation procedures for adoptively transferred cells by using a combination of bispecific antibodies directed to the EGF receptor (EGFR) on tumor cells and to CD3 and CD28 on T cells. Eleven patients with high-grade malignant glioma received 3 injections of 2 bispecific antibody fragments (EGFR x CD3 and EGFR x CD28) together with freshly isolated autologous lymphocytes via an Ommaya reservoir. Intracavitary fluid aspirated during immunotherapy was examined for markers of T-cell activation. Increased levels of soluble IL-2 receptor and TNF-alpha were detected in the intracavitary fluid of all patients tested. Two of the 11 treated patients experienced a beneficial response to therapy as defined by a transient contrast enhancement in subsequent MRI scans and prolonged survival. Side effects were transient and consisted of fever, nausea, headache and aggravation of pre-existing neurologic deficits. These adverse effects were most likely due to the antibody construct containing anti-CD3 specificity. Two patients developed cerebral edema and required steroid treatment.

Adult↗

PubMed2001

More hydrogen bonds for the (structural) biologist.

Why does a given protein structure form and why is this structure stable? These fundamental biochemical questions remain fascinating and challenging problems because the physical bases of the forces that govern protein structure, stability and folding are still not well understood. Now, a general concept of hydrogen bonding in proteins is emerging. This concept involves not only N-H and O-H donor groups, but also C-H, and not only N and O as acceptor groups, but also pi-systems. We postulate that the incorporation of the entirety of these interactions leads to a more complete description of the problem, and that this could provide new perspectives and possibly new answers.

Carrier Proteins↗

PubMed2001

Liposomes as drug carriers: a technological approach.

Liposome researchers have created a hugh variety of liposomal drug carriers in the past thirty years mainly by small-scale laboratory techniques using more or less well defined raw materials. Only a few of these liposomal preparations have made their way to approved drugs for clinical use in humans so far. The review gives a critical literature survey over key technologies, which are used to evaluate an appropriate lipid formula and to prepare, size, load and sterilise liposomes. It also deals with quality and shelf stability aspects of liposomal drug carriers.

Biotechnology↗

PubMed2001

Quantum-chemical analysis of C-H...O and C-H...N interactions in RNA base pairs--H-bond versus anti-H-bond pattern.

Geometries and interaction energies of unusual UU and AA base pairs with one standard hydrogen bond (H-bond) and additional C-H...O or C-H...N contacts have been determined by quantum-chemical methods taking into account electron correlation. Whereas the C-H bond length in the UU C-H...O contact increases upon complex formation (H-bond pattern), the C-H bond of the AA C-H....N interaction is shortened (anti-H-bond pattern). The same properties are found for model complexes between U or A and formaldehyde that have intermolecular C-H...acceptor contacts but no standard H-bonds. Both the C-H...acceptor H-bond and anti-H-bond interactions are attractive. A possible influence of the donor CH group charge distribution on the interaction pattern is discussed.

Adenine↗

PubMed2000

Effect of nucleoside analogues and oligonucleotides on hydrolysis of liposomal phospholipids.

The hydrolysis of the bilayer forming phospholipids resulting first of all in lysophospholipids and fatty acids is one limiting factor determining the shelf-life of liposomes. In several studies the influence of pH, buffer, lipid composition and other parameters on the hydrolysis of phospholipids have been demonstrated, but the influence of drugs has not yet been investigated systematically. In this study the influence of nucleoside analogues, especially 2', 2'-difluoro 2'-deoxycytidine (gemcitabine, dFdC) on the degradation of phospholipids was elucidated in more detail. It could be demonstrated that the interaction of dFdC with phospholipid bilayers promotes the hydrolysis of phospholipids in a concentration-dependent manner. Obviously two parts of the molecule, the amino group bound to the pyrimidine moiety and the 2', 2'-difluoro-2'-deoxyribose, seem to be responsible for the forced phospholipid hydrolysis. The dFdC-induced hydrolysis of phospholipids was influenced by pH, buffer, lipid composition and different anions. Optimization of the above parameters resulted in prolonged shelf-life of dFdC liposome dispersions, which is an important prerequisite for clinical practice.

Anions↗

PubMed1999

Role of target antigen in bispecific-antibody-mediated killing of human glioblastoma cells: a pre-clinical study.

Bispecific antibodies (bsAbs) directed to tumor-associated antigens and to receptors mediating T-cell activation, such as the TCR/CD3 complex and the co-stimulatory CD28 molecule, are capable of activating T cells at the surface of tumor cells, resulting in tumor-cell killing. Here we report the pre-clinical characterization of bispecific-antibody fragments (bsFab2) directed to 2 different glioblastoma-associated antigens: the EGF receptor (EGFR) and a chondroitin-sulfate proteoglycan (CSPG). Using cultured glioblastoma cells expressing both target antigens, we found that the ability of anti-tumor x anti-CD28 bsFab2 to mediate "targeted T-cell co-stimulation" is superior for constructs targeting the CSPG molecule, correlating with an approximately 6-fold higher expression level of this antigen on the cell surface. In contrast, bsFab2 triggering CD3 are more effective if they contain EGFR-target specificity. This indicates that the activity of anti-tumor x anti-CD3 constructs critically depends on properties of the antigen other than its expression level on the cell surface, e.g., its mobility in the membrane. These findings prompted us to use EGFR-targeting bsFab2 in an ongoing clinical trial with glioma patients.

Antibodies, Bispecific↗

PubMed1999

Tumor-growth inhibition with bispecific antibody fragments in a syngeneic mouse melanoma model: the role of targeted T-cell co-stimulation via CD28.

The ability of bispecific antibodies with anti-tumor x anti-CD3 specificity to mediate the killing of tumor cells by activated T cells has been demonstrated in many in vitro experiments. Moreover, long-term survival of lymphoma-bearing mice has been observed after treatment with such reagents. The therapeutic effect of bispecific antibodies in solid-tumor models has been less impressive, in particular if fragmented antibodies were used to avoid systemic T-cell activation by bispecific constructs binding to Fc-receptor-positive cells. Here we report that bispecific anti-tumor x anti-CD3-fragments markedly inhibit intraperitoneal as well as pulmonary tumor growth in mice inoculated with B16 melanoma cells, resulting in the long-term survival of animals. Therapeutic success critically depends on the number of recruitable effector cells at the site of tumor growth. A second bispecific construct triggering the co-stimulatory CD28-molecule on the T-cell surface increased tumor-cell killing in vitro and in vivo, despite rather low avidity of this reagent to mouse T cells. Finally, long-term-surviving animals showed improved survival after i.v. rechallenge with tumor cells, indicating that bispecific antibodies are capable of inducing long-lasting protective immunity.

Animals↗