Polymers, drug release, and drug-eluting stents

Implantable biomaterials mainly serve as physical support devices, carriers for bioactive molecules and guidance for tissue growth. For any application within or outside the cardiovascular area, biomaterials are subject to an extended set of requirements in order to establish safe application. These requirements mainly include acceptable biocompatibility and, if the material is to be degraded within the body, safe degradation characteristics. During degradation, biocompatible polymers are broken down into molecules that are metabolized and removed from the body via normal metabolic pathways. Major applications of these polymers include targeted drug delivery systems, resorbable sutures and orthopedic fixation devices. In the cardiovascular area they include biodegradable cardiovascular stents and drug-eluting stent (DES) coatings. This review focuses on general aspects of local drug delivery by implantable polymeric devices, with special emphasis on drug-eluting stents.     

Atp8b1 deficiency in mice reduces resistance of the canalicular membrane to hydrophobic bile salts and impairs bile salt transport

Progressive familial intrahepatic cholestasis type 1 (PFIC1, Byler disease, OMIM 211600) is a severe inherited liver disease caused by mutations in ATP8B1. ATP8B1 is a member of the type 4 subfamily of P-type ATPases, which are phospholipid flippases. PFIC1 patients generally develop end-stage liver disease before the second decade of life. The disease is characterized by impaired biliary bile salt excretion, but the mechanism whereby impaired ATP8B1 function results in cholestasis is unclear. In a mouse model for PFIC1, we observed decreased resistance of the hepatocanalicular membrane to hydrophobic bile salts as evidenced by enhanced biliary recovery of phosphatidylserine, cholesterol, and ectoenzymes. In liver specimens from PFIC1 patients, but not in those from control subjects, ectoenzyme expression at the canalicular membrane was markedly deficient. In isolated mouse livers Atp8b1 deficiency impaired the transport of hydrophobic bile salts into bile. In conclusion, our study shows that Atp8b1 deficiency causes loss of canalicular phospholipid membrane asymmetry that in turn renders the canalicular membrane less resistant toward hydrophobic bile salts. The loss of phospholipid asymmetry may subsequently impair bile salt transport and cause cholestasis.     

Endocrinology of reproduction and phase transition in locusts

In the last decade, important progress has been made in the experimental analysis of the endocrine mechanisms controlling reproduction and phase transition in locusts. Phase transition is a very fascinating, but complex, phenomenon of phenotypic plasticity that is triggered by changes in population density and can lead to the formation of extremely devastating hopper bands and adult gregarious locust swarms. While some phase characteristics change within hours, others appear more gradually in the next stage(s), or even in the next generation(s). In adults, the phase status also has a major influence on the process of reproduction. A better understanding of how solitarious locusts become gregarious and how this switch affects reproductive physiology may result in novel strategies to fight locust plagues. In this paper, we will review the current knowledge concerning this close interaction between locust phase polyphenism and reproduction.     

Microbial ecology of the closed artificial ecosystem MELiSSA (Micro-Ecological Life Support System Alternative): reinventing and compartmentalizing the Earth's food and oxygen regeneration system for long-haul space exploration missions

MELiSSA is a bioregenerative life support system designed by the European Space Agency (ESA) for the complete recycling of gas, liquid and solid wastes during long distance space exploration. The system uses the combined activity of different living organisms: microbial cultures in bioreactors, a plant compartment and a human crew. In this minireview, the development of a short-cut ecological system for the biotransformation of organic waste is discussed from a microorganism's perspective. The artificial ecological model--still in full development--that is inspired by Earth's own geomicrobiological ecosystem serves as an ideal study object on microbial ecology and will become an indispensable travel companion in manned space exploration.     

Mitochondrial adaptations within chronically ischemic swine myocardium

Experimental evidence has emerged that myocardial ischemic preconditioning can prime the mitochondria into a "stress-resistant state", so that cell death is reduced following prolonged severe ischemia and reperfusion. Using a swine model of chronically ischemic myocardium, we tested the hypothesis that mitochondria within the ischemic territory have also acquired a protective phenotype. Eleven swine underwent a left thoracotomy with placement of an external constrictor around the proximal left anterior descending (LAD) artery. By 10 weeks, a severe stenosis of the LAD artery was documented by quantitative coronary angiography (92 +/- 2%). Animals were sacrificed and myocardium was extracted from the LAD and remote regions. Mitochondria were isolated from subendocardium and subepicardium from LAD and remote regions and state 2 (substrate alone) and state 3 (+ADP) respiration were assessed with a Clark electrode. Within the LAD subendocardium, the respiratory control index was 2.68 +/- 0.17 and was lower than the remote subendocardium (3.64 +/- 0.08; P < 0.05). When exposed to 20 min anoxia with reoxygenation, the LAD region demonstrated a more preserved state 3 respiration compared with the remote region (99 +/- 14 versus 65 +/- 9 nmol O2/mg, respectively; P < 0.05). In parallel mitochondrial experiments, chemiluminescence was detected with the probe coelenterazine and superoxide generation in the LAD region in the presence of antimycin A was 574 +/- 108 RLU/30 s/microg and was nearly 50% lower than the remote region (979 +/- 175 RLU/30 s/microg; P < 0.05). Within the mitochondria, the expression of uncoupling protein (UCP) 2 by western gels was 20% higher in the LAD region compared with the remote region (P < 0.05) with no differences noted in UCP-3. In this swine model of chronic myocardial ischemia, isolated mitochondria from the ischemic tissue demonstrate preserved state 3 respiration following anoxia/reoxygenation, consistent with a stress-resistant state. This state is characterized by a mild degree of uncoupling under basal conditions and decreased superoxide generation. Uncoupling protein 2 expression is enhanced in the mitochondria, providing a potential mechanism for these favorable mitochondrial adaptations.     

Coding and non-coding polymorphisms in the lectin pathway activator L-ficolin gene in 188 Dutch blood bank donors

Human L-ficolin (FCN) is a serum lectin characterized by a collagen-like and a fibrinogen-like domain that can activate the lectin pathway of complement. Structural and functional similarities to mannose-binding lectin (MBL) suggest a role for L-ficolin in innate immunity. Structural polymorphisms in the MBL2 gene lead to functional deficiency of MBL. Polymorphisms in the FCN2 gene have not been studied previously. We developed 10 denaturing gradient gel electrophoresis (DGGE) assays to screen a total of 188 Dutch Caucasians for polymorphisms in FCN2. Total gene screening in this large cohort revealed 10 single nucleotide polymorphisms (SNPs). Interestingly, two conserved coding SNPs were found in exon 8, leading to amino acid substitutions within the fibrinogen-like domain. Fibrinogen-like domains are highly conserved among several proteins in many species. As this domain is responsible for binding of L-ficolin, these newly found coding polymorphisms could alter the affinity of the protein for its substrates and possibly alter the ability of L-ficolin to recognize invading microorganisms.     

Acute pulmonary embolism in childhood

Pulmonary embolism is an uncommon, but potentially fatal disease in children. Most children with pulmonary embolism have underlying clinical conditions, of which the presence of a central venous catheter is the most frequent. The clinical presentation is often subtle, or masked by the underlying clinical condition. Diagnostic as well as therapeutic strategies for pulmonary embolism in children are mostly extrapolated from studies in adults. Pulmonary angiography is still the gold standard in diagnosing pulmonary embolism, but several other radiographic tests can be used to diagnose pulmonary embolism, including ventilation-perfusion lung scanning, helical computed tomography, magnetic resonance imaging and echocardiography. The choice of treatment depends on the clinical presentation of the patient. Anticoagulation is the mainstay of therapy for children with pulmonary embolism. However, thrombolytic therapy can be considered for patients with hemodynamic instability. The outcome of pediatric pulmonary embolism is uncertain and needs to be studied.     

Tract-based diffusion tensor imaging in patients with schizophrenia and their non-psychotic siblings

Structural brain abnormalities have consistently been found in patients with schizophrenia. Diffusion tensor imaging (DTI) has been shown to be a useful method to measure white matter (WM) integrity in this illness, but findings in the earlier disease stages are inconclusive. Moreover, the relationship between WM microstructure and the familial risk for developing schizophrenia remains unresolved. From 126 patients with schizophrenia, 123 of their non-psychotic siblings and 109 healthy control subjects, DTI images were acquired on a 1.5 T MRI scanner. Mean fractional anisotropy (FA) was compared along averaged WM tracts, computed for the genu, splenium, left and right uncinate fasciculus, cingulum, inferior fronto-occipital fasciculus, fornix, arcuate fasciculus, and inferior longitudinal fasciculus. Fractional anisotropy (FA) was assessed for its unique environmental and familial (possibly heritable) aspects associated with schizophrenia, using structural equation modeling for these white matter tracts. The results of this study show that young adult (mean age 26.7 years) patients with schizophrenia did not differ in mean FA from healthy controls along WM fibers; siblings of patients showed higher mean FA in the left and right arcuate fasciculus as compared to patients and controls. With increasing age, an excessive decline in mean FA was found in patients as compared to siblings and healthy controls in the genu, left uncinate fasciculus, left inferior fronto-occipital fasciculus, and left inferior longitudinal fasciculus. Moreover, symptom severity was negatively correlated to mean FA in the arcuate fasciculus bilaterally in patients with schizophrenia. In young adult patients with schizophrenia integrity of individual tract-based (corticocortical) fibers can (still) be within normal limits. However, changes in the arcuate fasciculus may be relevant to (the risk to develop) psychosis, while a general and widespread loss of fiber integrity may be related to illness progression.     

Validation of the Transferrin Receptor for Drug Targeting to Brain Capillary Endothelial Cells In Vitro

Recently, we have shown that transferrin (Tf) is actively endocytosed by the Tf R on primary cultured bovine brain capillary endothelial cells (BCEC). The objective of this investigation is to determine whether the Tf R can facilitate endocytosis of a (protein) model drug, using Tf as a targeting vector. Secondly, the mechanism of endocytosis was investigated. Horseradish peroxidase (HRP, 40 kDa) was chosen as a model drug, since it normally does not cross the blood–brain barrier (BBB) and its concentration in biological media can be easily quantified.

Tf-HRP conjugates (1:1) are actively and specifically endocytosed by BCEC in vitro in a concentration and time-dependent manner. At an applied concentration of 3 μg/ml, association (a combination of binding and endocytosis) of Tf-HRP reached equilibrium at a concentration of 2 ng/mg cell protein after 1 h of incubation at 37°C. This was approximately 3-fold higher compared to binding at 4°C (0.6 ng/mg cell protein). Association of Tf-HRP was compared to BSA-HRP. After 2 h of incubation at 37°C association levels were 5.2 and 2.5 ng/mg cell protein, for Tf-HRP and BSA-HRP, respectively. Under those conditions, association of Tf-HRP could be inhibited to approximately 30% of total association by an excess of non-conjugated Tf, but not with BSA, while association of BSA-HRP could be inhibited by both proteins. Furthermore, by using specific inhibitors of endocytotic processes, it was shown that association of Tf-HRP is via clathrin-coated vesicles. Association of Tf-HRP is inhibited by phenylarsine oxide (an inhibitor of clathrin-mediated endocytosis) to 0.4 ng/mg cell protein, but not by indomethacin, which inhibits formation of caveolae. Finally, following iron scavenging by deferoxamine mesylate (DFO, resulting in a higher Tf R expression) a 5-fold increase in association of Tf-HRP to 15.8 ng/mg cell protein was observed.

In conclusion, the Tf R is potentially suitable for targeting of a (protein) cargo to the BBB and to facilitate its endocytosis by the BCEC.