Controlled delivery of 5-chlorouracil using poly(ortho esters) in filtering surgery for glaucoma.

PURPOSE: To evaluate the antimitotic and toxic effects of 5-chlorouracil (5-CU) and 5-fluorouracil (5-FU) and study their potential to delay filtering bleb closure in the rabbit eye when released by poly(ortho esters) (POE). METHODS: Rabbit Tenon fibroblasts and human conjunctival cells were incubated with various 5-CU and 5-FU concentrations. Antiproliferative effects and toxicity were evaluated at 24 and 72 hours by monotetrazolium, neutral red, and Hoechst tests and cell counting. Mechanisms of cell death were evaluated using TUNEL assay, annexin V binding, immunohistochemistry for anti-apoptosis-inducing factor (AIF) and LEI/L-DNase II. Trabeculectomy was performed in pigmented rabbits. Two hundred microliters of POE loaded with 1% wt/wt 5-FU or 5-CU was injected into the subconjunctival space after surgery. Intraocular pressure (IOP) and bleb persistence were monitored for 150 days. RESULTS: In vitro, 5-FU showed a higher antiproliferative effect and a more toxic effect than 5-CU. 5-FU induced cell necrosis, whereas 5-CU induced mostly apoptosis. The apoptosis induced by 5-CU was driven through a non-caspase-dependent pathway involving AIF and LEI/L-DNase II. In vivo, at 34 days after surgery, the mean IOP in the POE/5-CU-treated group was 83% of the baseline level and only 40% in the POE/5-FU-treated group. At 100 days after surgery, IOP was still decreased in the POE/5-CU group when compared with the controls and still inferior to the preoperative value. The mean long-term IOP, with all time points considered, was significantly (P < 0.0001) decreased in the POE/5-CU-treated group (6.0 +/- 2.4 mm Hg) when compared with both control groups, the trabeculectomy alone group (7.6 +/- 2.9 mm Hg), and the POE alone group (7.5 +/- 2.6 mm Hg). Histologic analysis showed evidence of functioning blebs in the POE-5-CU-treated eyes along with a preserved structure of the conjunctiva epithelium. CONCLUSIONS: The slow release of 5-CU from POE has a longstanding effect on the decrease of IOP after glaucoma-filtering surgery in the rabbit eye. Thus, the slow release of POE/5-CU may be beneficial for the prevention of bleb closure in patients who undergo complicated trabeculectomy.

What's so special about conversion disorder? A problem and a proposal for diagnostic classification.

Conversion disorder presents a problem for the revisions of DSM-IV and ICD-10, for reasons that are informative about the difficulties of psychiatric classification more generally. Giving up criteria based on psychological aetiology may be a painful sacrifice but it is still the right thing to do.

Fibrinmatrix erhöht Adhärenz von regenerativen peripheren Nervenzellen [Fibrin matrix enhances adherence of peripheral nerve regenerative cells]

Optimal seeding of a nerve conduit with cells is a core problem in tissue engineering of constructing an artificial nerve substitute to gap lesions in the peripheral nerve system. An ideal nerve gap substitute would have to present an equally distributed number of cells that can activate the regrowing axons. This work shows a new in vitro technique of two-step seeding of cells inside a conduit and on layered mats that allows a valuable targeting of the cells and a proven survival in the environment of poly-3-hydroxybutyrate (PHB) conduits. The technique uses two components of diluted fibrin glue Tisseel. Initially, the chosen area on the mat was coated with thrombin followed from the seeding of a fibrinogen-cell compound. Using Sprague Dawley rat cells, we could demonstrate with immunohistochemistry (S100, DAPI) techniques that undifferentiated (uMSC) and Schwann cells (SC) mimicking differentiated mesenchymal stem cells (dMSC) as well as SC can be suspended and targeted significantly better in dissolvable diluted fibrin glue than in growth medium. Analysis showed significantly better values for adherence (p < 0.001) and drop off (p < 0.05) from seeded cells. Using this two-step application allows the seeding of the cells to be more precise and simplifies the handling of cell transplantation.

Sublimation of new matrix candidates for high spatial resolution imaging mass spectrometry of lipids: enhanced information in both positive and negative polarities after 1,5-diaminonapthalene deposition.

Matrix sublimation has demonstrated to be a powerful approach for high-resolution matrix-assisted laser desorption ionization (MALDI) imaging of lipids, providing very homogeneous solvent-free deposition. This work presents a comprehensive study aiming to evaluate current and novel matrix candidates for high spatial resolution MALDI imaging mass spectrometry of lipids from tissue section after deposition by sublimation. For this purpose, 12 matrices including 2,5-dihydroxybenzoic acid (DHB), sinapinic acid (SA), α-cyano-4-hydroxycinnamic acid (CHCA), 2,6-dihydroxyacetphenone (DHA), 2',4',6'-trihydroxyacetophenone (THAP), 3-hydroxypicolinic acid (3-HPA), 1,8-bis(dimethylamino)naphthalene (DMAN), 1,8,9-anthracentriol (DIT), 1,5-diaminonapthalene (DAN), p-nitroaniline (NIT), 9-aminoacridine (9-AA), and 2-mercaptobenzothiazole (MBT) were investigated for lipid detection efficiency in both positive and negative ionization modes, matrix interferences, and stability under vacuum. For the most relevant matrices, ion maps of the different lipid species were obtained from tissue sections at high spatial resolution and the detected peaks were characterized by matrix-assisted laser desorption ionization time-of-flight/time-of-flight (MALDI-TOF/TOF) mass spectrometry. First proposed for imaging mass spectrometry (IMS) after sublimation, DAN has demonstrated to be of high efficiency providing rich lipid signatures in both positive and negative polarities with high vacuum stability and sub-20 μm resolution capacity. Ion images from adult mouse brain were generated with a 10 μm scanning resolution. Furthermore, ion images from adult mouse brain and whole-body fish tissue sections were also acquired in both polarity modes from the same tissue section at 100 μm spatial resolution. Sublimation of DAN represents an interesting approach to improve information with respect to currently employed matrices providing a deeper analysis of the lipidome by IMS.

Development of stable cell lines for production or regulated expression using matrix attachment regions.

One of the major hurdles of isolating stable, inducible or constitutive high-level producer cell lines is the time-consuming selection procedure. Given the variation in the expression levels of the same construct in individual clones, hundreds of clones must be isolated and tested to identify one or more with the desired characteristics. Various boundary elements (BEs), matrix attachment regions, and locus control regions (LCRs) were screened for their ability to augment the expression of heterologous genes in Chinese hamster ovary (CHO) cells. Of the chromatin elements assayed, the chicken lysozyme matrix-attachment region (MAR) was the only element to significantly increase stable reporter expression. We found that the use of the MAR increases the proportion of high-producing clones, thus reducing the number of clones that need to be screened. These benefits are observed both for constructs with MARs flanking the transgene expression cassette, as well as when constructs are co-transfected with the MAR on a separate plasmid. Moreover, the MAR was co-transfected with a multicomponent regulatable beta-galactosidase expression system in C2C12 cells and several clones exhibiting regulated expression were identified. Hence, MARs are useful in the development of stable cell lines for production or regulated expression.

Matrix-assisted laser desorption ionization-time of flight mass spectrometry as an alternative to 16S rRNA gene sequencing for identification of difficult-to-identify bacterial strains.

Conventional methods are sometimes insufficient to identify human bacterial pathogens, and alternative techniques, often molecular, are required. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) identified with a valid score 45.9% of 410 clinical isolates from 207 different difficult-to-identify species having required 16S rRNA gene sequencing. MALDI-TOF MS might represent an alternative to 16S rRNA gene sequencing.

Exhaled breath condensate as a matrix for combustion-based nanoparticle exposure and health effect evaluation

Health assessment and medical surveillance of workers exposed to combustion nanoparticles are challenging. The aim was to evaluate the feasibility of using exhaled breath condensate (EBC) from healthy volunteers for (1) assessing the lung deposited dose of combustion nanoparticles and (2) determining the resulting oxidative stress by measuring hydrogen peroxide (H2O2) and malondialdehyde (MDA). Methods: Fifteen healthy nonsmoker volunteers were exposed to three different levels of sidestream cigarette smoke under controlled conditions. EBC was repeatedly collected before, during, and 1 and 2 hr after exposure. Exposure variables were measured by direct reading instruments and by active sampling. The different EBC samples were analyzed for particle number concentration (light-scattering-based method) and for selected compounds considered oxidative stress markers. Results: Subjects were exposed to an average airborne concentration up to 4.3×10(5) particles/cm(3) (average geometric size ∼60-80 nm). Up to 10×10(8) particles/mL could be measured in the collected EBC with a broad size distribution (50(th) percentile ∼160 nm), but these biological concentrations were not related to the exposure level of cigarette smoke particles. Although H2O2 and MDA concentrations in EBC increased during exposure, only H2O2 showed a transient normalization 1 hr after exposure and increased afterward. In contrast, MDA levels stayed elevated during the 2 hr post exposure. Conclusions: The use of diffusion light scattering for particle counting proved to be sufficiently sensitive to detect objects in EBC, but lacked the specificity for carbonaceous tobacco smoke particles. Our results suggest two phases of oxidation markers in EBC: first, the initial deposition of particles and gases in the lung lining liquid, and later the start of oxidative stress with associated cell membrane damage. Future studies should extend the follow-up time and should remove gases or particles from the air to allow differentiation between the different sources of H2O2 and MDA.

Endothelial cells as key determinants of the tumor microenvironment: interaction with tumor cells, extracellular matrix and immune killer cells.

Besides tumor cells, the tumor microenvironment harbors a variety of host-derived cells, such as endothelial cells, fibroblasts, innate and adaptive immune cells. It is a complex and highly dynamic environment, providing very important cues to tumor development and progression. Tumor-associated endothelial cells play a key role in this process. On the one hand, they form tumor-associated (angiogenic) vessels through sprouting from locally preexisting vessels or recruitment of bone marrow-derived endothelial progenitor cells, to provide nutritional support to the growing tumor. On the other hand, they are the interface between circulating blood cells, tumor cells and the extracellular matrix, thereby playing a central role in controlling leukocyte recruitment, tumor cell behavior and metastasis formation. Hypoxia is a critical parameter modulating the tumor microenvironment and endothelial/tumor cell interactions. Under hypoxic stress, tumor cells produce factors that promote tumor angiogenesis, tumor cell motility and metastasis. Among these factors, VEGF, a main angiogenesis modulator, can also play a critical role in the control of immune tolerance. This review discusses some aspects of the role of endothelial cells within tumor microenvironment and emphasizes their interaction with tumor cells, the extracellular matrix and with immune killer cells. We will also address the role played by circulating endothelial progenitor cells and illustrate their features and mechanism of recruitment to the tumor microenvironment and their role in tumor angiogenesis.

Different transcriptional control of metabolism and extracellular matrix in visceral and subcutaneous fat of obese and rimonabant treated mice.

BACKGROUND: The visceral (VAT) and subcutaneous (SCAT) adipose tissues play different roles in physiology and obesity. The molecular mechanisms underlying their expansion in obesity and following body weight reduction are poorly defined. METHODOLOGY: C57Bl/6 mice fed a high fat diet (HFD) for 6 months developed low, medium, or high body weight as compared to normal chow fed mice. Mice from each groups were then treated with the cannabinoid receptor 1 antagonist rimonabant or vehicle for 24 days to normalize their body weight. Transcriptomic data for visceral and subcutaneous adipose tissues from each group of mice were obtained and analyzed to identify: i) genes regulated by HFD irrespective of body weight, ii) genes whose expression correlated with body weight, iii) the biological processes activated in each tissue using gene set enrichment analysis (GSEA), iv) the transcriptional programs affected by rimonabant. PRINCIPAL FINDINGS: In VAT, "metabolic" genes encoding enzymes for lipid and steroid biosynthesis and glucose catabolism were down-regulated irrespective of body weight whereas "structure" genes controlling cell architecture and tissue remodeling had expression levels correlated with body weight. In SCAT, the identified "metabolic" and "structure" genes were mostly different from those identified in VAT and were regulated irrespective of body weight. GSEA indicated active adipogenesis in both tissues but a more prominent involvement of tissue stroma in VAT than in SCAT. Rimonabant treatment normalized most gene expression but further reduced oxidative phosphorylation gene expression in SCAT but not in VAT. CONCLUSION: VAT and SCAT show strikingly different gene expression programs in response to high fat diet and rimonabant treatment. Our results may lead to identification of therapeutic targets acting on specific fat depots to control obesity.

A new poly(ortho ester)-based drug delivery system as an adjunct treatment in filtering surgery.

PURPOSE: Pharmacologic modulation of wound healing after glaucoma filtering surgery remains a major clinical challenge in ophthalmology. Poly(ortho ester) (POE) is a bioerodible and biocompatible viscous polymer potentially useful as a sustained drug delivery system that allows the frequency of intraocular injections to be reduced. The purpose of this study was to determine the efficacy of POE containing a precise amount of 5-fluorouracil (5-FU) in an experimental model of filtering surgery in the rabbit. METHODS: Trabeculectomy was performed in pigmented rabbit eyes. An ointmentlike formulation of POE containing 1% wt/wt 5-FU was injected subconjunctivally at the site of surgery, during the procedure. Intraocular pressure (IOP), bleb persistence, and ocular inflammatory reaction were monitored until postoperative day 30. Quantitative analysis of 5-FU was performed in the anterior chamber. Histologic analysis was used to assess the appearance of the filtering fistula and the polymer's biocompatibility. RESULTS: The decrease in IOP from baseline and the persistence of the filtering bleb were significantly more marked in the 5-FU-treated eyes during postoperative days 9 through 28. Corneal toxicity triggered by 5-FU was significantly lower in the group that received 5-FU in POE compared with a 5-FU tamponade. Histopathologic evaluation showed that POE was well tolerated, and no fibrosis occurred in eyes treated with POE containing 5-FU. CONCLUSIONS: In this rabbit model of trabeculectomy, the formulation based on POE and containing a precise amount of 5-FU reduced IOP and prolonged bleb persistence in a way similar to the conventional method of a 5-FU tamponade, while significantly reducing 5-FU toxicity.

Current cardiovascular risk management patterns with special focus on lipid lowering in daily practice in Switzerland.

Background: There may be a considerable gap between LDL cholesterol (LDL-C) and blood pressure (BP) goal values recommended by the guidelines and results achieved in daily practice. Design Prospective cross-sectional survey of cardiovascular disease risk profiles and management with focus on lipid lowering and BP lowering in clinical practice. Methods: In phase 1, the cardiovascular risk of patients with known lipid profile visiting their general practitioner was anonymously assessed in accordance to the PROCAM-score. In phase 2, high-risk patients who did not achieve LDL-C goal less than 2.6 mmol/l in phase 1 could be further documented. Results: Six hundred thirty-five general practitioners collected the data of 23 892 patients with known lipid profile. Forty percent were high-risk patients (diabetes mellitus or coronary heart disease or PROCAM-score >20%), compared with 27% estimated by the physicians. Goal attainment rate was almost double for BP than for LDL-C in high-risk patients (62 vs. 37%). Both goals were attained by 25%. LDL-C values in phase 1 and 2 were available for 3097 high-risk patients not at LDL-C goal in phase 1; 32% of patients achieved LDL-C goal of less than 2.6 mmol/l after a mean of 17 weeks. The most successful strategies for LDL-C reduction were implemented in only 22% of the high-risk patients. Conclusion: Although patients at high cardiovascular risk were treated more intensively than low or medium risk patients, the majority remained insufficiently controlled, which is an incentive for intensified medical education. Adequate implementation of Swiss and International guidelines would expectedly contribute to improved achievement of LDL-C and BP goal values in daily practice.

Interplay of oxidative, nitrosative/nitrative stress, inflammation, cell death and autophagy in diabetic cardiomyopathy.

Diabetes is a recognized risk factor for cardiovascular diseases and heart failure. Diabetic cardiovascular dysfunction also underscores the development of diabetic retinopathy, nephropathy and neuropathy. Despite the broad availability of antidiabetic therapy, glycemic control still remains a major challenge in the management of diabetic patients. Hyperglycemia triggers formation of advanced glycosylation end products (AGEs), activates protein kinase C, enhances polyol pathway, glucose autoxidation, which coupled with elevated levels of free fatty acids, and leptin have been implicated in increased generation of superoxide anion by mitochondria, NADPH oxidases and xanthine oxidoreductase in diabetic vasculature and myocardium. Superoxide anion interacts with nitric oxide forming the potent toxin peroxynitrite via diffusion limited reaction, which in concert with other oxidants triggers activation of stress kinases, endoplasmic reticulum stress, mitochondrial and poly(ADP-ribose) polymerase 1-dependent cell death, dysregulates autophagy/mitophagy, inactivates key proteins involved in myocardial calcium handling/contractility and antioxidant defense, activates matrix metalloproteinases and redox-dependent pro-inflammatory transcription factors (e.g. nuclear factor kappaB) promoting inflammation, AGEs formation, eventually culminating in myocardial dysfunction, remodeling and heart failure. Understanding the complex interplay of oxidative/nitrosative stress with pro-inflammatory, metabolic and cell death pathways is critical to devise novel targeted therapies for diabetic cardiomyopathy, which will be overviewed in this brief synopsis. This article is part of a Special Issue entitled: Autophagy and protein quality control in cardiometabolic diseases.