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Biomedical subjects

Stefan Fischer

Publications and source records attributed to Stefan Fischer.

At least 19 recordsLinked to original sources
PubMed2006

The preservation of phenotype and functionality of dendritic cells upon phagocytosis of polyelectrolyte-coated PLGA microparticles.

Biodegradable microparticles (MP) represent a promising and efficient delivery system for parenteral vaccination. Recently, MP have also been explored as tool for the ex vivo antigen loading of professional antigen-presenting cells such as dendritic cells (DC) to be used as cellular vaccines. The purpose of this study was to investigate various polycationic coatings on poly(lactide-co-glycolide) (PLGA) MP, with regard to their effect on phenotypic and functional maturation of monocyte-derived DC (MoDC) that had previously been loaded with the MP in vitro. The preparation and concomitant coating of the PLGA was performed by means of a solvent extraction/evaporation method using a recently developed microextrusion-based technique. The polyelectrolytes tested for MP coating encompassed aminodextran, chitosan, poly(ethylene imine) (PEI), poly(L-lysine) and protamine. Uncoated and differently coated PLGA MP were fed to immature MoDC, which ingested efficiently the different MP types irrespective of their surface coating. The MP-loaded immature MoDC were then matured with the help of a cytokine/PGE-2 maturation cocktail. Here, the presence of the ingested MP did not affect the MoDC maturation in terms of expression of the surface markers CD80, CD83, CD86, HLA-DR and MMR, irrespective of the MP surface coating. Importantly, none of the PLGA MP types alone induced significant maturation of MoDC in the absence of the maturation cocktail. MP-loaded and subsequently matured MoDC expressed high levels of the chemokine receptor CCR7, whose functional activity was evidenced by the migration of MoDC towards CCL21, irrespective of the presence of ingested MP. Further, MP-loaded and subsequently matured MoDC also secreted comparable amounts of IL-10 and IL-12p70, irrespective of the presence of ingested MP except for PEI-coated PLGA MP, which enhanced significantly the secretion of IL-12p70 in mature MoDC. In conclusion, phenotypic and functional maturation of MoDC by means of a maturation cocktail remained unchanged irrespective of the presence of previously ingested differently coated PLGA MP. This offers interesting perspectives for using these particulate systems together with entrapped antigens for ex vivo loading of MoDC in view of cellular immunotherapy.

Cell Proliferation↗
PubMed2006

Suppression of the back proton-transfer from Asp85 to the retinal Schiff base in bacteriorhodopsin: a theoretical analysis of structural elements.

The transfer of a proton from the retinal Schiff base to the nearby Asp85 protein group is an essential step in the directional proton-pumping by bacteriorhodopsin. To avoid the wasteful back reprotonation of the Schiff base from Asp85, the protein must ensure that, following Schiff base deprotonation, the energy barrier for back proton-transfer from Asp85 to the Schiff base is larger than that for proton-transfer from the Schiff base to Asp85. Here, three structural elements that may contribute to suppressing the back proton-transfer from Asp85 to the Schiff base are investigated: (i) retinal twisting; (ii) hydrogen-bonding distances in the active site; and (iii) the number and location of internal water molecules. The impact of the pattern of bond twisting on the retinal deprotonation energy is dissected by performing an extensive set of quantum-mechanical calculations. Structural rearrangements in the active site, such as changes of the Thr89:Asp85 distance and relocation of water molecules hydrogen-bonding to the Asp85 acceptor group, may participate in the mechanism which ensures that following the transfer of the Schiff base proton to Asp85 the protein proceeds with the subsequent photocycle steps, and not with back proton transfer from Asp85 to the Schiff base.

Animals↗
PubMed2006

Immune cells contribute to myelin degeneration and axonopathic changes in mice overexpressing proteolipid protein in oligodendrocytes.

Overexpression of the major myelin protein of the CNS, proteolipid protein (PLP), leads to late-onset degeneration of myelin and pathological changes in axons. Based on the observation that in white matter tracts of these mutants both CD8+ T-lymphocytes and CD11b+ macrophage-like cells are numerically elevated, we tested the hypothesis that these cells are pathologically involved in the primarily genetically caused neuropathy. Using flow cytometry of mutant brains, CD8+ cells could be identified as activated effector cells, and confocal microscopy revealed a close association of the T-cells with MHC-I+ (major histocompatibility complex class I positive) oligodendrocytes. Crossbreeding the myelin mutants with mice deficient in the recombination activating gene-1 (RAG-1) lacking mature T- and B-lymphocytes led to a reduction of the number of CD11b+ cells and to a substantial alleviation of pathological changes. In accordance with these findings, magnetic resonance imaging revealed less ventricular enlargement in the double mutants, partially because of more preserved corpora callosa. To investigate the role of CD8+ versus CD4+ T-lymphocytes, we reconstituted the myelin-RAG-1 double mutants with bone marrow from either CD8-negative (CD4+) or CD4-negative (CD8+) mice. The severe ventricular enlargement was only found when the double mutants were reconstituted with bone marrow from CD8+ mice, suggesting that the CD8+ lymphocytes play a critical role in the immune-related component of myelin degeneration in the mutants. These findings provide strong evidence that a primary glial damage can cause secondary immune reactions of pathological significance as it has been suggested for some forms of multiple sclerosis and other leukodystrophies.

Animals↗
PubMed2006

Analyzing large-scale structural change in proteins: comparison of principal component projection and Sammon mapping.

Effective analysis of large-scale conformational transitions in macromolecules requires transforming them into a lower dimensional representation that captures the dominant motions. Herein, we apply and compare two different dimensionality reduction techniques, namely, principal component analysis (PCA), a linear method, and Sammon mapping, which is nonlinear. The two methods are used to analyze four different protein transition pathways of varying complexity, obtained by using either the conjugate peak refinement method or constrained molecular dynamics. For the return-stroke in myosin, both Sammon mapping and PCA show that the conformational change is dominated by a simple rotation of a rigid body. Also, in the case of the T-->R transition in hemoglobin, both methods are able to identify the two main quaternary transition events. In contrast, in the cases of the unfolding transition of staphylococcal nuclease or the signaling switch of Ras p21, which are both more complex conformational transitions, only Sammon mapping is able to identify the distinct phases of motion.

Amino Acid Substitution↗
PubMed2006

Simulations of the myosin II motor reveal a nucleotide-state sensing element that controls the recovery stroke.

During the recovery stroke, the myosin motor is primed for the next power stroke by a 60 degree rotation of its lever arm. This reversible motion is coupled to the activation of the ATPase function of myosin through conformational changes along the relay helix, which runs from the Switch-2 loop near the ATP to the converter domain carrying the lever arm. Via a hydrogen bond between the side-chain of Asn475 on the relay helix and the Gly457/Ser456 peptide group on the Switch-2, the rotation of the converter domain is coupled to the formation of a hydrogen bond between Gly457 and gamma-phosphate that is essential for ATP hydrolysis. Here, molecular dynamics simulations of Dictyostelium discoideum myosin II in the two end conformations of the recovery stroke with different nucleotide states (ATP, ADP x Pi, ADP) reveal that the side-chain of Asn475 breaks away from Switch-2 upon ATP hydrolysis to make a hydrogen bond with Tyr573. This sensing of the nucleotide state is achieved by a small displacement of the cleaved gamma-phosphate towards Gly457 which in turn pushes Asn475 away. The sensing plays a dual role by (i) preventing the wasteful reversal of the recovery stroke while the nucleotide is in the ADP x Pi state, and (ii) decoupling the relay helix from Switch-2, thus allowing the power stroke to start upon initial binding to actin while Gly457 of Switch-2 keeps interacting with the Pi (known to be released only later after tight actin binding). A catalytically important salt bridge between Arg238 (on Switch-1) and Glu459 (on Switch-2), which covers the hydrolysis site, is seen to form rapidly when ATP is added to the pre-recovery stroke conformer and remains stable after the recovery stroke, indicating that it has a role in shaping the ATP binding site by induced fit.

Adenosine Diphosphate↗
PubMed2006

Insights into the chemomechanical coupling of the myosin motor from simulation of its ATP hydrolysis mechanism.

The molecular motor myosin converts chemical energy from ATP hydrolysis into mechanical work, thus driving a variety of essential motility processes. Although myosin function has been studied extensively, the catalytic mechanism of ATP hydrolysis and its chemomechanical coupling to the motor cycle are not completely understood. Here, the catalysis mechanism in myosin II is examined using quantum mechanical/molecular mechanical reaction path calculations. The resulting reaction pathways, found in the catalytically competent closed/closed conformation of the Switch-1/Switch-2 loops of myosin, are all associative with a pentavalent bipyramidal oxyphosphorane transition state but can vary in the activation mechanism of the attacking water molecule and in the way the hydrogens are transferred between the heavy atoms. The coordination bond between the Mg2+ metal cofactor and Ser237 in the Switch-1 loop is broken in the product state, thereby facilitating the opening of the Switch-1 loop after hydrolysis is completed, which is required for subsequent strong rebinding to actin. This reveals a key element of the chemomechanical coupling that underlies the motor cycle, namely, the modulation of actin unbinding or binding in response to the ATP or ADP x P(i) state of nucleotide-bound myosin.

Adenosine Triphosphate↗
PubMed2006

Structural and energetic determinants of primary proton transfer in bacteriorhodopsin.

In the light-driven bacteriorhodopsin proton pump, the first proton transfer step is from the retinal Schiff base to a nearby carboxylate group. The mechanism of this transfer step is highly controversial, in particular whether a direct proton jump is allowed. Here, we review the structural and energetic determinants of the direct proton transfer path computed by using a combined quantum mechanical/molecular mechanical approach. Both protein flexibility and electrostatic interactions play an important role in shaping the proton transfer energy profile. Detailed analysis of the energetics of putative transitions in the first half of the photocycle focuses on two elements that determine the likelihood that a given configuration of the active site is populated during the proton-pumping cycle. First, the rate-limiting barrier for proton transfer must be consistent with the kinetics of the photocycle. Second, the active-site configuration must be compatible with a productive overall pumping cycle.

Bacteriorhodopsins↗
PubMed2006

Glycine intravenous donor preconditioning is superior to glycine supplementation to low-potassium dextran flush preservation and improves graft function in a large animal lung transplantation model after 24 hours of cold ischemia.

OBJECTIVES: The potential role of glycine in combination with standard lung preservation with low-potassium dextran solution in lung ischemia-reperfusion injury has not been investigated in a preclinical porcine transplant model. METHODS: In a control group (n = 6), donor lungs were flushed with 1 liter of low-potassium dextran solution. In a second group (LPD-glyc, n = 6), low-potassium dextran solution was supplemented with 3.75 g of glycine. In a third group (IV-glyc, n = 6), donor preconditioning was performed by intravenous administration of 3.75 g glycine 1 hour before low-potassium dextran preservation. Grafts were stored in low-potassium dextran at 4 degrees C for 24 hours. Posttransplant graft function was assessed throughout a 7-hour observation period. RESULTS: In the control group, 2 recipients died of right-sided heart failure caused by severe ischemia-reperfusion injury. All animals of the glycine groups survived the entire observation period. Pulmonary vascular resistance remained significantly (P < .01) lower in both glycine groups when compared with controls. At the end of the observation period pulmonary vascular resistance in the control group was higher (P < .01) compared with the glycine groups (1310 +/- 319 dyn x sec x cm(-5) vs 879 +/- 127 dyn x sec x cm(-5) [LPD-glyc] vs 663 +/- 191 dyn x sec x cm(-5) [IV-glyc]). Changes of lung tissue water content were lower in the IV-glyc group compared with the LPD-control (P < .01) and LPD-glyc lungs (P < .05). Oxygenation (PO2/FiO2) was higher in the IV-glyc group compared with the LPD-glyc and control lungs (445 +/- 110 mm Hg vs 388 +/- 124 mm Hg [P < .01] vs 341 +/- 224 mm Hg [P < .001], respectively). DISCUSSION: Modification of low-potassium dextran solution with glycine or donor preconditioning ameliorates ischemia-reperfusion injury in lung transplantation. This intriguing approach merits further evaluation with respect to the mechanisms involved and may improve results in clinical lung preservation.

Animals↗
PubMed2006

Bridge to lung transplantation with the novel pumpless interventional lung assist device NovaLung.

BACKGROUND: Worsening of lung failure in patients awaiting a lung transplantation might lead to ventilation-refractory hypercapnia and respiratory acidosis. Most transplant centers consider pretransplantation extracorporeal membrane oxygenation as a contraindication for lung transplantation because of the poor outcome. We have, for the first time, applied the novel pumpless interventional lung assist NovaLung for bridge to lung transplantation in patients with severe ventilation-refractory hypercapnia. We report on our initial experience. METHODS: Between March 2003 and March 2005, 176 lung transplantations were performed, of which 60% were high-urgency lung transplantations. Twelve of the high-urgency recipients had severe ventilation-refractory hypercapnia and respiratory acidosis. These patients were connected to the novel pumpless interventional lung assist NovaLung for bridge to lung transplantation. RESULTS: The length of interventional lung assist NovaLung support was 15 +/- 8 days (4-32 days). PaO2, pH, and PaCO2 levels in arterial blood prior to interventional lung assist NovaLung implantation were 71 +/- 27 mm Hg, 7.121 +/- 0.1, and 128 +/- 42 mm Hg, respectively. Six hours after interventional lung assist NovaLung implantation, PaO2, pH, and PaCO2 levels had changed to 83 +/- 17 mm Hg (ns), 7.344 +/- 0.1 (P < .05), and 52 +/- 5 mm Hg (P < .05), respectively. Four patients died of multiorgan failure, 2 patients before and 2 after lung transplantation. Thus, 10 out of 12 patients were successfully bridged to lung transplantation, and 8 are still alive (1-year survival, 80%). CONCLUSIONS: This report suggests that interventional lung assist NovaLung implantation is an effective bridge to lung transplantation strategy in patients with ventilation-refractory hypercapnia.

Acidosis, Respiratory↗
PubMed2006

Long-term outcome after pulmonary retransplantation.

OBJECTIVE: Bronchiolitis obliterans syndrome has become the most limiting factor for long-term outcome after lung transplantation. Redo lung transplantation was performed for end-stage bronchiolitis obliterans syndrome. Long-term outcome was compared with that after primary lung transplantation as well as with other indications for retransplantation. METHODS: Of 614 lung transplantation procedures performed at our institution, 54 (8.5%) were redo transplants. These were stratified into different groups according to the indication for redo transplantation, including chronic graft failure/bronchiolitis obliterans syndrome, acute graft failure, and posttransplantation airway complications. Long-term survival was compared with that of the primary lung transplantation cohort, thereby respecting the need for pretransplant mechanical ventilatory support in a subanalysis. In addition, recurrence of bronchiolitis obliterans syndrome after redo lung transplantation was compared with the occurrence of bronchiolitis obliterans after primary transplantation. RESULTS: A 1-year survival of 50% was achieved after redo lung transplantation for acute graft failure and airway complications as well as after primary lung transplantation in patients with pretransplant ventilatory support. Retransplantation for bronchiolitis obliterans syndrome revealed superior 1- (78%) and 5-year (62%) survivals, which were not different from those of first-time lung transplant recipients. In addition, we found a similar incidence of bronchiolitis syndrome after retransplantation for BOS compared with its occurrence after primary lung transplantation. CONCLUSION: Redo lung transplantation for end-stage bronchiolitis obliterans syndrome leads to acceptable long-term outcome in selected patients. Future analyses of redo lung transplantation data should generally stratify bronchiolitis obliterans syndrome from other indications with higher mortality.

Adolescent↗
PubMed2006

Role of immune cells in animal models for inherited peripheral neuropathies.

Mice expressing half of the normal dose of protein zero (P0+/- mice) or completely deficient gap-junction protein connexin 32 -/- mice mimic demyelinating forms of inherited neuropathies, such as Charcot-Marie-Tooth (CMT) neuropathies type 1B and CMT type 1X, respectively. In both models, an almost normal myelin formation is observed during the first months of life, followed by a slowly progressing demyelinating neuropathy. In both models, there is a substantial increase of CD8+ T-lymphocytes and macrophages within the demyelinating nerves. Recently, this has also been observed in mice mildly overexpressing human peripheral myelin protein 22 kD mimicking the most common form of CMT, CMT type 1A. In all demyelinating models, the macrophages show close contacts with intact myelin sheaths or demyelinated axons, suggesting an active role of these cells in myelin degeneration. Additionally, fibroblast-like cells contact macrophages, suggesting a functional role of fibroblast-like cells in macrophage activation. By cross-breeding P0+/- and gap-junction protein connexin 32-/- mice with immunodeficient recombination activating gene-1-deficient mutants, a substantial alleviation of the demyelinating phenotype was observed. Similarly, cross-breeding of P0+/- mice with mutants with a defect in macrophage activation led to an alleviated phenotype as well. These findings demonstrate that the immune system is involved in the pathogenesis of demyelinating neuropathies. In contrast, in P0-/- mice, which display a compromised myelin compaction and axonal loss from onset, immune cells appear to have a neuroprotective effect because cross-breeding with recombination activating gene-1 mutants leads to an aggravation of axonopathic changes. In the present review, we discuss the influence of the immune system on inherited de- and dysmyelination regarding disease mechanisms and possible clinical implications.

Animals↗
PubMed2006

Implicit learning -- explicit knowing: a role for sleep in memory system interaction.

There is evidence that sleep supports the enhancement of implicit as well as explicit memories (i.e., two memory systems that during learning normally appear to act together). Here, employing a serial reaction time task (SRTT) paradigm, we examined the question whether sleep can provide explicit knowledge on an implicitly acquired skill. At learning, young healthy subjects (n = 20) were first trained on the SRTT. Then, implicit knowledge was assessed on two test blocks, in which grammatically incorrect target positions were occasionally interspersed by the difference in reaction times between grammatically correct and incorrect target positions. To assess explicit sequence knowledge, thereafter subjects performed on a generation task in which they were explicitly instructed to predict the sequential target positions. In half the subjects, learning took place before a 9-hour retention interval filled with nocturnal sleep (sleep group), in the other half, the retention interval covered a 9-hour period of daytime wakefulness (wake group). At subsequent retesting, both testing on the generation task and the SRTT test blocks was repeated. At learning before the retention interval, subjects displayed significant implicit sequence knowledge which was comparable for the sleep and wake groups. Moreover, both groups did not display any explicit sequence knowledge as indicated by a prediction performance not differing from chance on the generation task. However, at retesting, there was a distinct gain in explicit knowledge in the subjects who had slept in the retention interval, whereas generation task performance in the wake group remained at chance level. SRTT performance in the test blocks at retesting did not indicate any further gain in skill (i.e., unchanged reaction time differences between grammatically correct and incorrect target positions) independently of whether subjects had slept or remained awake after learning. Our results indicate a selective enhancement of explicit memory formation during sleep. Because before sleep subjects only had implicit knowledge on the sequence of target transitions, these data point to an interaction between implicit and explicit memory systems during sleep-dependent off-line learning.

Adolescent↗
PubMed2005

One-step preparation of polyelectrolyte-coated PLGA microparticles and their functionalization with model ligands.

This work aimed at the development of a novel surfactant-free, one-step process for the concomitant formation of poly(lactide-co-glycolide) (PLGA) microparticles (MP) and surface coating with the polyelectrolyte chitosan, which is suitable for subsequent covalent conjugation of bioactive ligands. The technology is based on solvent extraction from an O/W-dispersion using a static micromixer. Surface coating occurred through interaction of the negatively charged, nascent PLGA MP with the polycationic chitosan, which was dissolved in the aqueous extraction fluid. Particles of 1-10 mum in diameter were produced with excellent reproducibility. The chitosan-coated PLGA MP were spherical and showed a smooth surface without pores, as demonstrated by scanning electron microscopy (SEM). The chitosan coatings were characterized by zeta potential measurements and X-ray photoelectron spectroscopy (XPS). The functional amino groups of chitosan were used to conjugate two model ligands to the coating, i.e. fluorescamine and NHS-PEG-biotin. The presence of the conjugated ligands was revealed by confocal laser scanning microscopy (CLSM) and fluorescence activated cell sorting (FACS). Evidence for biotinylation was demonstrated through binding of fluorescently labelled streptavidin. The developed platform technology is straightforward and flexible. Future studies will focus on the design of microparticulate carriers with bioactive surfaces, e.g. as antigen delivery systems.

Biocompatible Materials↗
PubMed2005

Motor memory consolidation in sleep shapes more effective neuronal representations.

Learning a motor skill involves a latent process of consolidation that develops after training to enhance the skill in the absence of any practice and crucially depends on sleep. Here, we show that this latent consolidation during sleep changes the brain representation of the motor skill by reducing overall the neocortical contributions to the representation. Functional magnetic resonance brain imaging was performed during initial training and 48 h later, at retesting, on a sequential finger movement task with training followed by either a night of regular sleep or sleep deprivation. An additional night of sleep for all subjects served to rule out unspecific effects of sleep loss at retrieval testing. Posttraining sleep, but not sleep deprivation, led to improved motor skill performance at retrieval. This sleep-dependent improvement was linked to greatly reduced brain activation in prefrontal, premotor, and primary motor cortical areas, along with a stronger involvement of left parietal cortical regions. Our findings indicate that storing a motor skill during sleep reorganizes its brain representation toward enhanced efficacy.

Adolescent↗
PubMed2005

Health-related quality of life in two hundred-eighty lung transplant recipients.

BACKGROUND: Health-related quality of life (HRQoL) has increasingly been accepted as a supplementary outcome measure for patients after lung transplantation (LTx). METHODS: Using a retrospective cross-sectional study design, 280 LTx recipients (3 months to 14 years after LTx) were asked to assess their HRQoL using a generic (Quality of Life Profile for Chronic Diseases) questionnaire, which was previously validated for this specific population. In addition, the questionnaire was also performed by 155 healthy participants. RESULTS: All sub-scale findings ranged from 2.40 to 3.08 (0 to 4) for all patients after LTx. A significantly reduced HRQoL was reported by the sub-cohort of patients living 5 to 6 years with the allograft for all sub-scales (p < 0.006), except for Social Functioning, and was associated with the incidence of bronchiolitis obliterans syndrome (BOS; p < 0.05). Cystic fibrosis patients (p < 0.05), single-lung transplant recipients (p < 0.05) and patients of older age (p < 0.05) showed significantly decreased physical ability ratings. Patients who remained free of infection and late acute rejection episodes scored themselves significantly higher with regard to their Relaxation Capabilities (p < 0.05 for rejection; p < 0.05 for infection) and Social Functioning (p < 0.01 for rejection; p < 0.05 for infection) vs patients who experienced infection or rejection episodes. Comparisons with a normative cohort showed similar HRQoL scales for LTx patients and the healthy population, except with regard to Social Functioning (p < 0.01). CONCLUSIONS: Self-ratings for HRQoL were high for all dimensions for the entire sample, and remained relatively similar even for medium- and long-term survivors. HRQoL was dependent on incidence of infections, rejections and the onset of BOS. Despite differences in life expectancy of LTx patients compared with the healthy population, HRQoL self-ratings were within similar ranges.

Adolescent↗