ABSTRACT
Red-light responsiveness of photoswitches is a highly desired property for many important application areas such as biology or material sciences. The main approach to elicit this property uses strategic substitution of long-known photoswitch motives such as azobenzenes or diarylethenes. Only very few photoswitches possess inherent red-light absorption of their core chromophore structures. Here, we present a strategy to convert the long-known purple indirubin dye into a prolific red-light-responsive photoswitch. In a supramolecular approach, its photochromism can be changed from a negative to a positive one, while at the same time, significantly higher yields of the metastable E-isomer are obtained upon irradiation. E- to Z-photoisomerization can then also be induced by red light of longer wavelengths. Indirubin therefore represents a unique example of reversible photoswitching using entirely red light for both switching directions.
ABSTRACT
BMP2 and TGFß1 are functional antagonists of pathological remodeling in the arteries, heart, and lung; however, the mechanisms in VSMCs, and their disturbance in pulmonary arterial hypertension (PAH), are unclear. We found a pro-proliferative TGFß1-Stat3-FoxO1 axis in VSMCs, and PPARγ as inhibitory regulator of TGFß1-Stat3-FoxO1 and TGFß1-Smad3/4, by physically interacting with Stat3 and Smad3. TGFß1 induces fibrosis-related genes and miR-130a/301b, suppressing PPARγ. Conversely, PPARγ inhibits TGFß1-induced mitochondrial activation and VSMC proliferation, and regulates two glucose metabolism-related enzymes, platelet isoform of phosphofructokinase (PFKP, a PPARγ target, via miR-331-5p) and protein phosphatase 1 regulatory subunit 3G (PPP1R3G, a Smad3 target). PPARγ knockdown/deletion in VSMCs activates TGFß1 signaling. The PPARγ agonist pioglitazone reverses PAH and inhibits the TGFß1-Stat3-FoxO1 axis in TGFß1-overexpressing mice. We identified PPARγ as a missing link between BMP2 and TGFß1 pathways in VSMCs. PPARγ activation can be beneficial in TGFß1-associated diseases, such as PAH, parenchymal lung diseases, and Marfan's syndrome.
Subject(s)
Bone Morphogenetic Protein 2/metabolism , Cell Proliferation , Glucose/metabolism , Myocytes, Smooth Muscle/cytology , PPAR gamma/metabolism , Signal Transduction , Transforming Growth Factor beta1/metabolism , Animals , Cells, Cultured , Female , Male , Mice, Inbred C57BL , Muscle, Smooth, Vascular/cytology , Muscle, Smooth, Vascular/metabolism , Myocytes, Smooth Muscle/metabolism , Pulmonary Artery/cytology , Pulmonary Artery/metabolismABSTRACT
SCOPE: Isoflavones are discussed to improve serum lipoproteins and body composition and to reduce cardiovascular disease risk in postmenopausal women (PMW). LDL receptors (LDLR) and scavenger receptor CD36 (CD36) play a pivotal role in the regulation of plasma LDL-cholesterol concentrations (LDL-chol). We investigated the impact of isoflavones on the receptor expression of both receptors in leukocytes of PMW. METHODS AND RESULTS: A randomized, double-blind, placebo-controlled trial in parallel design was conducted to assess the effects of an isoflavone-enriched soy extract (117.4 mg/day isoflavone aglycone equivalents) for 12 weeks on serum LDL-chol, LDLR, and CD36 expression on leukocytes in 170 healthy PMW. Baseline and after 12 weeks, blood lipid concentrations, anthropometric data and body composition were determined. Receptor expression on leukocytes was measured by means of flow cytometry. After the intervention, no significant differences were found for LDLR and CD36 expression on leukocytes. A significant increase of serum LDL-chol was shown for the isoflavone group (p = 0.03) after 12 weeks. Body fat content and VAT were not affected. CONCLUSION: Isoflavone supplementation for 12 weeks did not change LDLR and CD36 expression on leukocytes of PMW and did not affect body fat content and visceral adipose tissue (VAT), but slightly increased serum LDL-chol.
Subject(s)
CD36 Antigens/blood , Isoflavones/pharmacology , Postmenopause , Receptors, LDL/blood , Aged , Body Composition/drug effects , Dietary Supplements , Double-Blind Method , Female , Humans , Leukocytes/drug effects , Leukocytes/metabolism , Lipids/blood , Lymphocytes/drug effects , Lymphocytes/metabolism , Middle Aged , Monocytes/drug effects , Monocytes/metabolism , Postmenopause/drug effects , Glycine max/chemistryABSTRACT
Dormancy in non-sporulating bacteria is an interesting and underexplored phenomenon with significant medical implications. In particular, latent tuberculosis may result from the maintenance of Mycobacterium tuberculosis bacilli in non-replicating states in infected individuals. Uniquely, growth of M. tuberculosis in aerobic conditions in potassium-deficient media resulted in the generation of bacilli that were non-culturable (NC) on solid media but detectable in liquid media. These bacilli were morphologically distinct and tolerant to cell-wall-targeting antimicrobials. Bacterial counts on solid media quickly recovered after washing and incubating bacilli in fresh resuscitation media containing potassium. This resuscitation of growth occurred too quickly to be attributed to M. tuberculosis replication. Transcriptomic and proteomic profiling through adaptation to, and resuscitation from, this NC state revealed a switch to anaerobic respiration and a shift to lipid and amino acid metabolism. High concordance with mRNA signatures derived from M. tuberculosis infection models suggests that analogous NC mycobacterial phenotypes may exist during disease and may represent unrecognized populations in vivo. Resuscitation of NC bacilli in potassium-sufficient media was characterized by time-dependent activation of metabolic pathways in a programmed series of processes that probably transit bacilli through challenging microenvironments during infection.
Subject(s)
Anti-Infective Agents/chemistry , Mycobacterium tuberculosis/physiology , Potassium/chemistry , Bacterial Proteins/chemistry , Electrophoresis, Gel, Two-Dimensional , Gene Expression Regulation, Bacterial , Humans , Mass Spectrometry , Phenotype , Phosphotransferases (Alcohol Group Acceptor)/chemistry , Proteome , Proteomics , RNA, Messenger/metabolism , Stem Cells , TranscriptomeABSTRACT
BACKGROUND: Illuminating the role of the microtubule-associated protein tau in neurodegenerative diseases is of increasing importance, supported by recent studies establishing novel functions of tau in synaptic signalling and cytoskeletal organization. In severe dementias like Alzheimer's disease (AD), synaptic failure and cognitive decline correlate best with the grade of tau-pathology. To address synaptic alterations in tauopathies, we analyzed the effects of mutant tau expression on excitatory postsynapses in vivo. RESULTS: Here we followed the fate of single dendritic spines in the neocortex of a tauopathy mouse model, expressing human P301S mutated tau, for a period of two weeks. We observed a continuous decrease in spine density during disease progression, which we could ascribe to a diminished fraction of gained spines. Remaining spines were enlarged and elongated, thus providing evidence for morphological reorganization in compensation for synaptic dysfunction. Remarkably, loss of dendritic spines in cortical pyramidal neurons occurred in the absence of neurofibrillary tangles (NFTs). Therefore, we consider prefibrillar tau species as causative for the observed impairment in spine plasticity. CONCLUSIONS: Dendritic spine plasticity and morphology are altered in layer V cortical neurons of P301S tau transgenic mice in vivo. This does not coincide with the detection of hyperphosphorylated tau in dendritic spines.
Subject(s)
Cerebral Cortex/physiopathology , Dendritic Spines/physiology , Neuronal Plasticity/physiology , Tauopathies/physiopathology , tau Proteins/genetics , tau Proteins/metabolism , Animals , Cerebral Cortex/pathology , Dendritic Spines/pathology , Disease Models, Animal , Immunohistochemistry , Male , Mice, Transgenic , Microscopy, Confocal , Mutation , Pyramidal Cells/pathology , Pyramidal Cells/physiopathology , Tauopathies/geneticsABSTRACT
Contact freezing has long been discussed as a candidate for cloud ice formation at temperatures warmer than about -25 degrees C, but until now the molecular mechanism underlying this process has remained obscure and little quantitative information about the size and temperature dependent contact freezing properties of the various aerosol species is available. In this contribution, we present the first quantitative measurements of the freezing probability of a supercooled droplet upon a single contact with a size selected illite mineral particle. It is found that this probability is a strong function of temperature and aerosol particle size. For the particles investigated and on the minute time scale of the experiment, contact freezing indeed dominates immersion freezing for all temperatures.
ABSTRACT
Tauopathies are widespread neurodegenerative disorders characterised by the intracellular accumulation of hyperphosphorylated tau. Especially in Alzheimer's disease, pathological alterations in the retina are discussed as potential biomarkers to improve early diagnosis of the disease. Using mice expressing human mutant P301S tau, we demonstrate for the first time a straightforward optical approach for the in vivo detection of fibrillar tau in the retina. Longitudinal examinations of individual animals revealed the fate of single cells containing fibrillar tau and the progression of tau pathology over several months. This technique is most suitable to monitor therapeutic interventions aimed at reducing the accumulation of fibrillar tau. In order to evaluate if this approach can be translated to human diagnosis, we tried to detect fibrillar protein aggregates in the post-mortem retinas of patients that had suffered from Alzheimer's disease or Progressive Supranuclear Palsy. Even though we could detect hyperphosphorylated tau, we did not observe any fibrillar tau or Aß aggregates. In contradiction to previous studies, our observations do not support the notion that Aß or tau in the retina are of diagnostic value in Alzheimer's disease.
Subject(s)
Retina/metabolism , Tauopathies/metabolism , tau Proteins/metabolism , Adult , Aged , Aged, 80 and over , Alzheimer Disease/genetics , Alzheimer Disease/metabolism , Alzheimer Disease/pathology , Animals , Disease Models, Animal , Female , Humans , Male , Mice , Mice, Transgenic , Microscopy, Confocal , Middle Aged , Phosphorylation , Retina/pathology , Tauopathies/genetics , Tauopathies/pathology , tau Proteins/geneticsABSTRACT
Ablation of a cochlea causes total sensory deafferentation of the cochlear nucleus in the brainstem, providing a model to investigate nervous degeneration and formation of new synaptic contacts in the adult brain. In a quantitative electron microscopical study on the plasticity of the central auditory system of the Wistar rat, we first determined what fraction of the total number of synaptic contact zones (SCZs) in the anteroventral cochlear nucleus (AVCN) is attributable to primary sensory innervation and how many synapses remain after total unilateral cochlear ablation. Second, we attempted to identify the potential for a deafferentation-dependent synaptogenesis. SCZs were ultrastructurally identified before and after deafferentation in tissue treated for ethanolic phosphotungstic acid (EPTA) staining. This was combined with pre-embedding immunocytochemistry for gephyrin identifying inhibitory SCZs, the growth-associated protein GAP-43, glutamate, and choline acetyltransferase. A stereological analysis of EPTA stained sections revealed 1.11±0.09 (S.E.M.)×10(9) SCZs per mm(3) of AVCN tissue. Within 7 days of deafferentation, this number was down by 46%. Excitatory and inhibitory synapses were differentially affected on the side of deafferentation. Excitatory synapses were quickly reduced and then began to increase in number again, necessarily being complemented from sources other than cochlear neurons, while inhibitory synapses were reduced more slowly and continuously. The result was a transient rise of the relative fraction of inhibitory synapses with a decline below original levels thereafter. Synaptogenesis was inferred by the emergence of morphologically immature SCZs that were consistently associated with GAP-43 immunoreactivity. SCZs of this type were estimated to make up a fraction of close to 30% of the total synaptic population present by ten weeks after sensory deafferentation. In conclusion, there appears to be a substantial potential for network reorganization and synaptogenesis in the auditory brainstem after loss of hearing, even in the adult brain.
Subject(s)
Auditory Pathways/physiopathology , Cochlear Nucleus/physiology , Synapses/metabolism , Afferent Pathways/physiopathology , Animals , Brain Stem/physiopathology , GAP-43 Protein/metabolism , Hearing Loss/physiopathology , Microscopy, Electron , Rats , Rats, WistarABSTRACT
The protein kinase inhibitor staurosporine is one of the most potent and frequently used proapoptotic stimuli, although its mechanism of action is poorly understood. Here, we show that staurosporine as well as its analog 7-hydroxystaurosporine (UCN-01) not only trigger the classical mitochondrial apoptosis pathway but, moreover, activate an additional novel intrinsic apoptosis pathway. Unlike conventional anticancer drugs, staurosporine and UCN-01 induced apoptosis in a variety of tumor cells overexpressing the apoptosis inhibitors Bcl-2 and Bcl-x(L). Furthermore, activation of this novel intrinsic apoptosis pathway by staurosporine did not rely on Apaf-1 and apoptosome formation, an essential requirement for the mitochondrial pathway. Nevertheless, as demonstrated in caspase-9-deficient murine embryonic fibroblasts, human lymphoma cells, and chicken DT40 cells, staurosporine-induced apoptosis was essentially mediated by caspase-9. Our results therefore suggest that, in addition to the classical cytochrome c/Apaf-1-dependent pathway of caspase-9 activation, staurosporine can induce caspase-9 activation and apoptosis independently of the apoptosome. Since staurosporine derivatives have proven efficacy in clinical trials, activation of this novel pathway might represent a powerful target to induce apoptosis in multidrug-resistant tumor cells.
Subject(s)
Antineoplastic Agents/pharmacology , Apoptosis/drug effects , Apoptotic Protease-Activating Factor 1/metabolism , Caspase 9/metabolism , Staurosporine/pharmacology , Apoptosis/physiology , Apoptotic Protease-Activating Factor 1/genetics , Cell Line, Tumor , Cytochromes c/genetics , Cytochromes c/metabolism , Gene Deletion , Gene Expression Regulation, Neoplastic/drug effects , Humans , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Staurosporine/analogs & derivatives , bcl-X Protein/genetics , bcl-X Protein/metabolismABSTRACT
Alzheimer's disease (AD) is the most common cause of cognitive decline in the elderly and is characterized by massive neuronal loss in the brain. Stem cell factor (SCF) is a hematopoietic growth factor that promotes neuroprotective effects and supports neurogenesis in the brain. Decreased SCF plasma levels have been described in AD patients. Whether SCF plasma levels are also associated with the rate of cognitive decline in AD patients has not been reported so far. In the present study, we demonstrate that SCF plasma levels are significantly decreased in AD patients with fast cognitive decline (decrease of Mini-Mental State Examination [MMSE] score > 4 after one year; n = 12) compared to AD patients with slow cognitive decline (decrease of MMSE score ≤ 4 after one year; n = 28) (fast versus slow cognitive decline: mean ± SD: 1051.1 ± 178.7 versus 1237.9 ± 274.2 pg/ml; p = 0.037). Moreover, SCF plasma levels correlated with the rate of cognitive decline after one year follow-up period (r = 0.315; p = 0.048). In a multiple linear regression analysis, independent predictors of the rate of cognitive decline in our study cohort were age, MMSE scores at baseline, SCF plasma levels, as well as brain-derived neurotrophic factor and activated glycoprotein (GP) IIb/IIIa. These results suggest that lower SCF plasma levels are associated with a higher rate of cognitive decline in AD patients. Thus, treatment strategies increasing SCF plasma levels could be useful for delaying the progression of AD. Further prospective studies are needed to elucidate the value of plasma SCF in a multimarker approach determining AD prognosis.
Subject(s)
Alzheimer Disease/blood , Alzheimer Disease/complications , Cognition Disorders/etiology , Stem Cell Factor/blood , Aged , Aged, 80 and over , Alzheimer Disease/pathology , Brain/pathology , Cognition Disorders/blood , Enzyme-Linked Immunosorbent Assay/methods , Female , Follow-Up Studies , Humans , Male , Mental Status Schedule , Neuropsychological Tests , Regression AnalysisABSTRACT
Phenotypic and genotypic diversifications of Pseudomonas aeruginosa in the airways of patients with cystic fibrosis (CF) promote long-term survival of bacteria during chronic lung infection. Twelve clonally related, sequential mucoid and non-mucoid paired P. aeruginosa isolates obtained from three Danish CF patients were investigated. The in vitro biofilm formation capacity was studied under static and flow through conditions and the global gene expression profiles were investigated by Affymetrix GeneChip. Regulatory genes of alginate production and quorum sensing (QS) system were sequenced and measurements of the alginate production and the detection of the QS signal molecules were performed. Comparisons of mucoid and non-mucoid isolates from early and late stages of the infection showed that the mucoid phenotype maintained over a decade the capacity to form in vitro biofilm and showed an unaltered transcriptional profile, whereas substantial alterations in the transcriptional profiles and loss of the capacity to form in vitro biofilms were observed in corresponding isolates of the non-mucoid phenotype. The conserved gene expression pattern in the mucoid isolates vs the diversity of changes in non-mucoid isolates observed in this particular P. aeruginosa clone reflects different adaptation strategies used by these two phenotypes in the different niches of the CF lung environment.
Subject(s)
Cystic Fibrosis/microbiology , Lung Diseases/microbiology , Pseudomonas Infections/microbiology , Pseudomonas aeruginosa , Alginates , Bacterial Proteins/genetics , Bacterial Proteins/physiology , Biofilms , Chronic Disease , Cystic Fibrosis/complications , Denmark , Gene Expression Profiling , Genes, Bacterial , Genotype , Glucuronic Acid/biosynthesis , Glucuronic Acid/genetics , Hexuronic Acids , Humans , Lung Diseases/etiology , Mutation , Oligonucleotide Array Sequence Analysis , Phenotype , Pseudomonas Infections/etiology , Pseudomonas aeruginosa/genetics , Pseudomonas aeruginosa/isolation & purification , Pseudomonas aeruginosa/physiology , Quorum Sensing/genetics , Quorum Sensing/physiology , Sigma Factor/genetics , Sigma Factor/physiology , Trans-Activators/genetics , Trans-Activators/physiologyABSTRACT
An ideal diagnostic test for Alzheimer's disease (AD) should be non-invasive and easily applicable. Thus, there is a clear need to search for biomarkers in blood. In the present study, we have used multivariate data analysis [support vector machine (SVM)] to investigate whether a blood-based biomarker panel allows discrimination between AD patients and healthy controls at the individual level. We collected a total of 155 serum samples from individuals with early AD and age-matched healthy controls and measured serum levels of 24 markers involved in several biological pathways by ELISA. The dataset was randomly split into a training set for predictor discovery and classification training and a test set for class prediction of blinded samples (3:1 ratio) to evaluate the chosen predictors and parameters. After selection of a feature group of the three most discriminative parameters (cortisol, von Willebrand factor, oxidized LDL antibodies) in the training set, the application of SVM to the training/independent test dataset resulted in an 81.7%/87.1% correct classification for AD and control subjects. In conclusion, we identified a panel of three blood markers, which allowed SVM-based distinguishing of AD patients from healthy controls on a single-subject classification level with clinically relevant accuracy and validity. Blood-based biomarkers might have utility in AD diagnostics as screening tool before further classification with CSF biomarkers and imaging. Future studies should examine whether blood-based biomarkers may also be useful to differentiate AD patients from other dementias.
Subject(s)
Alzheimer Disease/blood , Alzheimer Disease/diagnosis , Aged , Aged, 80 and over , Alzheimer Disease/immunology , Autoantibodies/analysis , Biomarkers/blood , Cohort Studies , Enzyme-Linked Immunosorbent Assay , Female , Humans , Hydrocortisone/blood , Lipoproteins, LDL/antagonists & inhibitors , Male , Middle Aged , Multivariate Analysis , Sensitivity and Specificity , Support Vector Machine , von Willebrand Factor/analysisABSTRACT
The manipulation of gene expression by RNA interference could play a key role in future neurotherapies, for example in the development of biohydrid implants to bridge nerve and spinal cord lesion gaps. Such resorbable biomaterial prostheses could serve as growth substrates together with specific siRNA to foster neuronal regeneration. To the best of our knowledge, we are the first to biofunctionalize neuronal prostheses with siRNA. We analyzed neuronal and Schwann cell responses to scrambled siRNA coated polydioxanone polymer filaments designed to imitate pro-regenerative bands of Büngner for oriented axonal regrowth. With a view to future clinical applications we were especially interested in potentially detrimental side effects. We employed a variety of in vitro methods, including a novel impedance electrode microchamber assay, fluorescence and scanning electron microscopy, metabolic labeling and RT-PCR. We found that the application of chitosan/siRNA nanoparticles (1) did not affect glial cell motility or (2) axonal growth in contrast to other formulations, (3) only slightly reduced proliferation, and (4) did not induce inflammatory responses that might hamper axonal regeneration. The data suggest that chitosan/siRNA nanoparticle-coated polymer filaments are suitable for use in biohybrid implants with no significant side effects on neuronal and glial cells.
Subject(s)
Axons/physiology , Neurons/physiology , RNA, Small Interfering/administration & dosage , Analysis of Variance , Animals , Biocompatible Materials , Chitosan , Immunohistochemistry , Nanoparticles , RNA, Small Interfering/genetics , Rats , Rats, Inbred Lew , Schwann Cells/physiologyABSTRACT
The neurotrophin brain-derived neurotrophic factor (BDNF) plays a critical role in neuronal survival, synaptic plasticity, and memory. Several recent studies have demonstrated altered BDNF serum levels in Alzheimer's disease (AD) patients. However, the association of BDNF serum levels with the rate of cognitive decline in AD patients is still unclear. We demonstrate that BDNF serum levels are significantly decreased in AD patients with fast cognitive decline [decrease of Mini-Mental State Examination (MMSE) score >4/yr; n=12] compared to AD patients with slow cognitive decline (decrease of MMSE score ≤4/yr, n=28) and show a significant correlation with the rate of cognitive decline during 1 yr follow-up. These results suggest that higher BDNF serum levels are associated with a slower rate of cognitive decline in AD patients. Further longitudinal studies are necessary to elucidate the kinetics and the potential role of serum BDNF as a surrogate marker of disease progression in AD patients.
Subject(s)
Alzheimer Disease/physiopathology , Brain-Derived Neurotrophic Factor/blood , Cognition Disorders/physiopathology , Cognition , Biomarkers , Disease Progression , Female , Humans , Male , Neuropsychological Tests , Time FactorsABSTRACT
The pathology of Alzheimer's disease (AD) is characterized by the accumulation of amyloid-ß (Aß) peptide, hyperphosphorylated tau protein, neuronal death, and synaptic loss. By means of long-term two-photon in vivo imaging and confocal imaging, we characterized the spatio-temporal pattern of dendritic spine loss for the first time in 3xTg-AD mice. These mice exhibit an early loss of layer III neurons at 4 months of age, at a time when only soluble Aß is abundant. Later on, dendritic spines are lost around amyloid plaques once they appear at 13 months of age. At the same age, we observed spine loss also in areas apart from amyloid plaques. This plaque independent spine loss manifests exclusively at dystrophic dendrites that accumulate both soluble Aß and hyperphosphorylated tau intracellularly. Collectively, our data shows that three spatio-temporally independent events contribute to a net loss of dendritic spines. These events coincided either with the occurrence of intracellular soluble or extracellular fibrillar Aß alone, or the combination of intracellular soluble Aß and hyperphosphorylated tau.
Subject(s)
Alzheimer Disease/pathology , Amyloid beta-Protein Precursor/physiology , Presenilin-1/physiology , tau Proteins/physiology , Alzheimer Disease/genetics , Alzheimer Disease/metabolism , Amino Acid Substitution , Amyloid beta-Protein Precursor/genetics , Amyloid beta-Protein Precursor/metabolism , Animals , Brain Diseases/genetics , Brain Diseases/metabolism , Brain Diseases/pathology , Dendritic Spines/genetics , Dendritic Spines/metabolism , Dendritic Spines/pathology , Female , Frontal Lobe/metabolism , Frontal Lobe/pathology , Hippocampus/metabolism , Hippocampus/pathology , Immunohistochemistry , Male , Mice , Mice, Transgenic , Microscopy, Confocal , Mutation , Neurons/metabolism , Neurons/pathology , Plaque, Amyloid/genetics , Plaque, Amyloid/metabolism , Plaque, Amyloid/pathology , Presenilin-1/genetics , Presenilin-1/metabolism , Somatosensory Cortex/metabolism , Somatosensory Cortex/pathology , Time Factors , tau Proteins/genetics , tau Proteins/metabolismABSTRACT
Nerve guide implants approved for human application in the peripheral nervous system generally fail to bridge lesion gaps longer than 2 cm and cannot match the clinical performance of autologous nerve transplants. Since current synthetic implants are simply hollow tubes, we aim to recreate the native microarchitecture of nerves inside the tubular implants. Most importantly, in the regenerating nerve, dedifferentiated Schwann cells align to form thousands of long glial strands, which act as guiding structures for the regrowing axons. In order to artificially induce the formation of Schwann cell strands, 28 µm thick, endless poly-p-dioxanone filaments (PDO) were synthesized with longitudinal grooves. A polycationic coating on the PDO filaments rendered the polymer surface cell-permissive and induced the growth of highly oriented Schwann cells with polarized expression of N-cadherin at cell-cell contact sites. In vitro cell proliferation on three-dimensional PDO filaments was significantly increased in comparison to planar PDO substrates. Time lapse video recordings revealed high Schwann cell motility, which is advantageous for the repopulation of cell-free implants after implantation. In a pilot study we employed a novel microsurgical technique in vivo. All axon fascicles were selectively dissected from sciatic rat nerves, and the remaining epineural tube was filled with hundreds of PDO filaments. Histological analysis 6 weeks postoperatively showed no fibrosis or encapsulation but instead longitudinal cell alignment and axonal regrowth. The data suggest that the addition of microstructured PDO filaments to the lumen of synthetic tubular implants might significantly improve performance.
Subject(s)
Bioengineering/methods , Nerve Regeneration/physiology , Schwann Cells/physiology , Sciatic Neuropathy/physiopathology , Sciatic Neuropathy/surgery , Animals , Biocompatible Materials/therapeutic use , Bromodeoxyuridine/metabolism , Cell Movement/physiology , Cell Proliferation , Dioxanes/therapeutic use , Disease Models, Animal , Female , Indoles , Polymers/therapeutic use , Rats , Rats, Inbred Lew , Schwann Cells/transplantation , Time Factors , Video Recording/methodsABSTRACT
Alzheimer's disease (AD) can be treated with inhibitors of the enzyme acetylcholinesterase (AChE). There is evidence that AChE inhibitors promote neuroprotective effects and neurogenesis in the central nervous system (CNS). However, the mechanisms by which AChE inhibitors mediate these effects are still not well understood. One possible mechanism could be the up-regulation of haematopoietic growth factors (HGFs), also known to promote neuroprotective effects and to stimulate neurogenesis in the CNS. In the present study we investigated the impact of a 15-month treatment with the AChE inhibitor donepezil on blood levels of the HGFs stem cell factor (SCF), stromal cell-derived factor 1 (SDF-1), granulocyte colony- stimulating factor (G-CSF) and vascular endothelial growth factor (VEGF) in 19 patients with AD and 45 age-matched healthy controls. Before treatment with donepezil we found in AD patients significantly decreased SCF plasma concentrations (661.1+/-40.0 pg/ml) compared to healthy controls (997.7+/-33.7 pg/ml, p<0.001) but no significant differences between both groups concerning blood levels of SDF-1, G-CSF and VEGF. After 15 months' treatment SCF plasma levels increased significantly in the AD patients (764.5+/-41.5 pg/ml, p=0.016). In addition, we found a significant positive correlation between SCF plasma levels at baseline and changes of cognitive functions over the 15-month period (r=0.521, p=0.022). For the other HGFs we were unable to show a significant impact of donepezil treatment. Our findings indicate that donepezil treatment of AD patients is associated with an up-regulation of SCF plasma levels, which may contribute to neuroprotection and neurogenesis in the CNS.
Subject(s)
Alzheimer Disease/blood , Alzheimer Disease/drug therapy , Indans/therapeutic use , Piperidines/therapeutic use , Stem Cell Factor/blood , Aged , Alzheimer Disease/diagnosis , Biomarkers/blood , Brief Psychiatric Rating Scale , Donepezil , Female , Humans , Male , Time Factors , Treatment OutcomeABSTRACT
The consequences of O-acetylated alginate-producing Pseudomonas aeruginosa biofilms in the lungs of chronically infected cystic fibrosis (CF) patients are tolerance to both antibiotic treatments and effects on the innate and the adaptive defense mechanisms. In clinical trials, azithromycin (AZM) has been shown to improve the lung function of CF patients. The present study was conducted in accordance with previous in vitro studies suggesting that the effect of AZM may be the inhibition of alginate production, blockage of quorum sensing (QS), and increased sensitivity to hydrogen peroxide and the complement system. Moreover, we show that AZM may affect the polymerization of P. aeruginosa alginate by the incomplete precipitation of polymerized alginate and high levels of readily dialyzable uronic acids. In addition, we find that mucoid bacteria in the stationary growth phase became sensitive to AZM, whereas cells in the exponential phase did not. Interestingly, AZM-treated P. aeruginosa lasI mutants appeared to be particularly resistant to serum, whereas bacteria with a functional QS system did not. We show in a CF mouse model of chronic P. aeruginosa lung infection that AZM treatment results in the suppression of QS-regulated virulence factors, significantly improves the clearance of P. aeruginosa alginate biofilms, and reduces the severity of the lung pathology compared to that in control mice. We conclude that AZM attenuates the virulence of P. aeruginosa, impairs its ability to form fully polymerized alginate biofilms, and increases its sensitivity to complement and stationary-phase killing, which may explain the clinical efficacy of AZM.
Subject(s)
Alginates/metabolism , Anti-Bacterial Agents/pharmacology , Azithromycin/pharmacology , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis Transmembrane Conductance Regulator/physiology , Lung Diseases/drug therapy , Lung Diseases/microbiology , Pseudomonas Infections/drug therapy , Pseudomonas Infections/microbiology , Pseudomonas aeruginosa/drug effects , Quorum Sensing/drug effects , Animals , Anti-Infective Agents, Local/pharmacology , Dose-Response Relationship, Drug , Female , Humans , Hydrogen Peroxide/pharmacology , Lung/microbiology , Lung/pathology , Lung Diseases/pathology , Male , Mice , Mice, Knockout , Virulence Factors/metabolismABSTRACT
SLH domains (for surface layer homology) are involved in the attachment of proteins to bacterial cell walls. The data presented here assign the conserved TRAE motif within SLH domains a key role for the binding. The charged amino acids arginine (R) or/and glutamic acid (E) were replaced via site-directed mutagenesis by different amino acids. Effects were visualized in an in vitro binding assay using native cell wall sacculi of Thermoanaerobacterium thermosulfurigenes EM1 and different variants of an SLH protein which consisted of the triplicate SLH domain of xylanase XynA of this bacterium and which was purified after expression in Escherichia coli. The results indicated (1) that the TRAE motif is critical for the binding function of SLH domains, (2) that a functional TRAE motif is necessary in all three domains, (3) that a least one (preferentially positively) charged amino acid in the TRAE motif is required for the functionality of the SLH domain, and (4) that the position of the negatively and positively charged amino acids is important. The finding that the cell wall of T. thermosulfurigenes EM1 contains pyruvate (4 microg mg(-1)) is in agreement with the hypothesis that pyruvylated secondary cell wall polymers function as ligand for SLH domains.
Subject(s)
Bacterial Adhesion , Bacterial Proteins/chemistry , Cell Wall/metabolism , Membrane Proteins/chemistry , Thermoanaerobacterium/physiology , Amino Acid Sequence , Arginine/genetics , Bacterial Adhesion/genetics , Bacterial Proteins/genetics , Binding Sites , Conserved Sequence , Glutamic Acid/genetics , Membrane Proteins/genetics , Mutagenesis, Site-Directed , Protein Structure, Tertiary , Recombinant Proteins/genetics , Thermoanaerobacterium/classification , Thermoanaerobacterium/geneticsABSTRACT
Pseudomonas aeruginosa causes a chronic infection in the lungs of cystic fibrosis (CF) patients by establishing an alginate-containing biofilm. The infection has been studied in several animal models; however, most of the models required artificial embedding of the bacteria. We present here a new pulmonary mouse model without artificial embedding. The model is based on a stable mucoid CF sputum isolate (NH57388A) with hyperproduction of alginate due to a deletion in mucA and functional N-acylhomoserine lactone (AHL)-based quorum-sensing systems. Chronic lung infection could be established in both CF mice (Cftr(tmlUnc-/-)) and BALB/c mice, as reflected by the detection of a high number of P. aeruginosa organisms in the lung homogenates at 7 days postinfection and alginate biofilms, surrounded by polymorphonuclear leukocytes in the alveoli. In comparison, both an AHL-producing nonmucoid revertant (NH57388C) from the mucoid isolate (NH57388A) and a nonmucoid isolate (NH57388B) deficient in AHL were almost cleared from the lungs of the mice. This model, in which P. aeruginosa is protected against the defense system of the lung by alginate, is similar to the clinical situation. Therefore, the mouse model provides an improved method for evaluating the interaction between mucoid P. aeruginosa, the host, and antibacterial therapy.