Confidence Interval Constraint-Based Regularization Framework for PET Quantization.

In this paper, a new generic regularized reconstruction framework based on confidence interval constraints for tomographic reconstruction is presented. As opposed to usual state-of-the-art regularization methods that try to minimize a cost function expressed as the sum of a data-fitting term and a regularization term weighted by a scalar parameter, the proposed algorithm is a two-step process. The first step concentrates on finding a set of images that rely on the direct estimation of confidence intervals for each reconstructed value. Then, the second step uses confidence intervals as a constraint to choose the most appropriate candidate according to a regularization criterion. Two different constraints are proposed in this paper. The first one has the main advantage of strictly ensuring that the regularized solution will respect the interval-valued data-fitting constraint, thus preventing over-smoothing of the solution while offering interesting properties in terms of spatial and statistical bias/variance trade-off. Another regularization proposition based on the design of a smoother constraint also with appealing properties is proposed as an alternative. The competitiveness of the proposed framework is illustrated in comparison to other regularization schemes using analytical and GATE-based simulation and real PET acquisition.

[Ocular Hypotension: How the Retina Surgeon Sees the Causes and Therapeutic Options]. / Okuläre Hypotonie - Ursachen und therapeutische Möglichkeiten aus Sicht des Netzhautchirurgen.

Ocular hypotension is a result of a lack of production or a loss of intraocular fluid. Intraocular inflammation, drugs, or proliferative vitreoretinopathy (PVR) with overgrowth of the ciliary body can result in reduced secretion of intraocular fluid. Loss of intraocular fluid can result from external loss, such as in fistulating surgery or trauma, or internally, e.g. from cyclodialysis clefts or retinal detachment. In this review, we discuss the causal therapy of ocular hypotension: fixation of the ciliary body, removal of ciliary body membranes, surgery for PVR, choice of tamponade, possibilities and limitations of an iris diaphragm, and pharmacological options.

[About the Effects of VEGF-A Antagonists on Molecular and Cellular Level]. / Die Wirkung der VEGF-A-Antagonisten auf molekularer und zellulärer Ebene.

Anti-VEGF-A therapy is successfully established as a routine therapy to treat wet age-related macular degeneration. Indications have been extended to other retinal diseases. Three different substances have been demonstrated to be active. However, the efficacy of these substances is highly variable in heterogeneous groups of patients and may include non-responders and relapses, so that there may be very individual treatment effects. It is speculated that differences in the molecular properties or structures of the three substances might explain these observations. This article therefore summarises the recent publications on this topic and discusses their relevance. Apart from common features such as VEGF-A affinity, the substances exhibit differences, including the stability of the VEGF-A/molecule complexes and the ability to neutralise angiogenic molecules other than only VEGF-A. At the cellular level, a variety of different methods have been used and the results are often inconsistent. It is therefore not yet possible to predict the clinical properties of VEGF-A neutralising substances on the basis of their known molecular properties or cellular effects.

Acute obturator internus muscle strain in a rugby player: a case report.

A 28-year-old male rugby player presented with severe onset of right hip pain when he fell awkward after a ruck during an international match. A rare case of an acute strain of the obturator internus muscle, a deep muscle of the hip joint, is reported, which resolved completely after a period of rest and intense active physical therapy.

Ocular delivery systems for poorly soluble drugs: an in-vivo evaluation.

For highly potent but poorly water-soluble drugs like cyclosporine A, the development of aqueous formulations providing an increase of corneal drug tissue levels, and thus of bioavailability, to increase patient compliance is still a challenge. Therefore, we designed two water-based liquid application systems, an in-situ nanosuspension (INS) and a micellar solution (MS), and tested both formulations in vivo at the rabbit cornea for tolerability and the tissue uptake of CsA. The evaluation of the biological tolerability by periodical eye examination during 180 min and quantification in a defined grading system revealed that the INS evoked minimal to no irritations whereas the MS was perfectly tolerated. After the observation period, the rabbits were sacrificed and the corneal tissue levels of CsA were analyzed. The INS and the MS both showed high levels of 1683±430 ngCsA/gcornea and 826±163 ngCsA/gcornea, respectively, and exceeded drug tissue levels reported for Restasis(®) (350 ngCsA/gcornea) and cationic emulsions (750 ngCsA/gcornea). These results marked our INS and MS as outstanding novel approaches for the treatment of inflammatory corneal diseases.

[An analysis of the pathophysiology of inherited diseases]. / Über die Klärung pathopysiologischer Ursachen genetisch bedingter Erkrankungen.

Evidence-based ophthalmology relies on a knowledge of the pathophysiological mechanisms at the molecular level. An example is the anti-VEGF therapy to treat the wet form of age-related macular degeneration. Its therapeutic effect is due to the neutralisation of a single type of molecule, the VEGF-A. The analysis of pathophysiological mechanisms of inherited diseases represents a unique opportunity to develop precise molecular therapeutic approaches. This analysis begins with the identification of the responsible gene which is followed by investigation of the function of its gene product along with the mutation-dependent changes using animal models and investigation of single molecules in expression systems. In this process the investigation of the pathomechanisms plays a central role. This review provides an orientation about studies on the pathophysiology of inherited diseases with a description of its methods and basic pathomechanisms. Furthermore, examples will be given on how the analysis of inherited retinal diseases has led to an understanding of the pathomechanisms of acquired diseases such as age-dependent macular degeneration and to the development of new therapeutic approaches.

[The role of retinal pigment epithelium in visual functions]. / Die Rolle des retinalen Pigmentepithels im Rahmen visueller Funktionen.

The evolution of light sensitive cells probably began with a primitive functional unit composed of a photoreceptor cell and a pigmented cell. Even during embryonic development this functional unit is formed in a differentiation process in which the two interacting partners depend on each other. For some of the most important forms of retinal degeneration this knowledge on the functional cooperation between retinal pigment epithelium and photoreceptors is of great importance for analysis and development of therapeutic approaches. In this way mutations of genes which are expressed in photoreceptors can lead to diseases which start in the retinal pigment epithelium and vice versa. This article summarizes the variety of different functions of the retinal pigment epithelium and describes the failure of those functions which are of most clinical importance.

[Oxidative stress at the retinal pigment epithelium - experimental implications for protection]. / Oxidativer Stress am retinalen Pigmentepithel - experimentelle Ansätze zur Protektion.

Oxidative stress at the retinal pigment epithelium (RPE) is involved in the pathophysiology of age-related macula degeneration (ARMD). Observations on a clinical or laboratory level have revealed that supplementation of antioxidative scavengers failed in many cases. A potential therapeutic target is the cellular signal transduction cascade initiated by oxidative stress which results, e. g., in altered expression of pro- and antiagiogenic factors as well as induction of apoptosis. This review summarises the current literature on cellular effects of free radicals and deduces potential therapeutic approaches to protect the RPE from oxidative damage.

Antiapoptotic properties of erythropoietin: novel strategies for protection of retinal pigment epithelial cells.

INTRODUCTION: Oxidative damage of the retinal pigment epithelium (RPE) may play a role in the development and progression of age-related macula degeneration (ARMD). Therapeutic reduction of oxidative stress failed or had only slight effects in ARMD patients. This study evaluates antiapoptotic properties of erythropoietin (epo) at the RPE as a novel approach to protect RPE cells against oxidative damage. MATERIALS AND METHODS: Cultured ARPE-19 cells were exposed to hydroxyl (OH) radicals generated from H(2)O(2) under catalysis of Fe(3+) (Fenton reaction) for 5 min. Apoptosis rate was determined by Annexin V labelling and terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling assay. Epo was added in concentrations from 0 to 100 U/ml to the media 24 and 1 h before radical exposure as well as shortly after radical exposure. Expression of epo receptor was determined by western blotting. RESULTS: Hydroxyl radical exposure induced an increase of apoptosis rate from virtually 0 to 11.8+/-1.7%. Apoptosis was detectable up to 24 h after radical exposure and reached its maximum after 6 h. Epo reduced apoptosis rate by up to 88% even if applied after the radical exposure. Best protection was achieved at 5 U/ml epo. Western blot confirmed presence of epo receptor independent of a pre-incubation of the cells with epo. DISCUSSION: Epo exerts antiapoptotic effects on cultured RPE cells even if applied after the radical exposure. This might qualify epo as future candidate for therapy and prevention of dry ARMD.

Spread of a low-fitness drug-resistant Mycobacterium tuberculosis strain in a setting of high human immunodeficiency virus prevalence.

The fitness cost associated with the evolution of resistance to rifampin in Mycobacterium tuberculosis may be different in clinical isolates compared to in vitro-generated mutants. An atypical Beijing strain (attenuated phenotype) demonstrated the ability to spread despite acquiring resistance to rifampin. Transmission was linked to human immunodeficiency virus coinfection (P = 0.029), raising concern for the spread of drug resistance in vulnerable populations.

An outbreak of drug-resistant tuberculosis caused by a Beijing strain in the western Cape, South Africa.

During October 2005, four children in a school in Cape Town were identified with multidrug-resistant tuberculosis (MDR-TB). Genetic analysis confirmed that these isolates belonged to a single cluster (Beijing cluster 220) and that all harboured a -15 inhA(C-T) promoter mutation demonstrating transmission. Genetic analysis of isolates cultured from patients from the Boland-Overberg-South Cape-Karoo and Cape Town regions showed that 28% (58/209) of patients infected with a Beijing strain had the cluster 220 genotypes and that all harboured the same -15 inhA(C-T) promoter mutation. The presence of these transmissible MDR-TB strains may pose a threat to the community, and rigorous infection control measures are needed to ensure the safety of those exposed.

[Function of bestrophin]. / Funktion des Bestrophins.

Clarification of the function of bestrophin, the gene product of VMD2, establishes a basis for the understanding of the pathomechanisms leading to Best's vitelliform macular degeneration. Studies of heterologously expressed bestrophin showed that bestrophin can function as a Cl(-) channel. All four known bestrophins were found to display Cl(-) channel activity. A loss in Cl(-) channel function would elegantly explain the development of the leading symptom for Best's disease, the reduction of the light peak amplitude in the patient's electro-oculogram. However, there are still gaps in the chain of evidence demonstrating that bestrophin is a Cl(-) channel, and this hypothesis is inconsistent with newly published follow-up observations. In an alternative hypothesis bestrophin appears as a regulator of voltage-dependent Ca(2+) channels assuming an indirect involvement of bestrophin in the generation of the light peak. Further studies on either bestrophin-deficient mice or transgenic mice will show that either one of the hypotheses is right or maybe both will be proven correct, showing bestrophin as a Cl(-) channel and Ca(2+) channel regulator.

Flufenamic acid enhances current through maxi-K channels in the trabecular meshwork of the eye.

PURPOSE: Flufenamic acid relaxes trabecular meshwork, a smooth muscle-like tissue involved in the regulation of ocular outflow in the eye. In this study, we attempted to determine if ionic channels are involved in this response. METHODS: Cultured human (HTM) and bovine (BTM) trabecular meshwork cells were investigated using the patch-clamp technique. RESULTS: In trabecular meshwork, flufenamic acid (10(-5) M) reversibly stimulated outward current to 406 +/- 71% of initial outward current level in BTM (n = 10) and 294 +/- 75% of initial current level in HTM (n = 12) in all cells investigated; no significant differences emerged. The response was dosage-dependent. Replacement of potassium in all solutions eliminated the response to flufenamic acid (n = 4, BTM). Blocking K(ATP ) channels with glibenclamide (10(-5) M, n = 6) and small-conductance calcium-activated potassium channels with apamin (10(-6) M, n = 5) had no effect. A direct effect on calcium channels could also not be detected. Blockage of the large-conductance calcium-activated potassium channel (maxi-K) by iberiotoxin (10(-7) M) suppressed 87 +/- 9% (n = 6; HTM) and 91 +/- 10% (n = 6; BTM) of the response. Depleting the cells of calcium did not significantly alter the response to flufenamic acid. CONCLUSIONS: Flufenamic acid stimulates maxi-K channels in trabecular meshwork of both human and bovine origin. This should lead to hyperpolarization, closure of L-type channels and lowered cytosolic calcium levels, possibly explaining the relaxation observed in response to this substance.

Muscarinic receptors of the M2 subtype in human and bovine trabecular meshwork.

BACKGROUND: The trabecular meshwork is a tissue actively involved in the regulation of intraocular pressure via contractile mechanisms. The present study was performed to investigate the effects of muscarinic m2-receptor antagonists on trabecular meshwork contractility and to identify the m2 muscarinic receptor in human and bovine trabecular meshwork cells. METHODS: Isometric tension measurements of bovine trabecular meshwork strips were performed using a custom-made force length transducer. Western blot and immunoprecipitation analysis was used to detect the m2-receptor proteins in membrane preparations of human and bovine trabecular meshwork cells. RESULTS: Immunoblotting results showed the expression of an m2-receptor protein band at 56 kDa in both human and bovine trabecular meshwork cells. Two different m2-receptor antagonists were tested on trabecular meshwork contractility. After carbachol-induced contraction (10(-6) M set to 100% contractile force), specific m2-receptor antagonists were applied. 3 alpha-Chloroimperaline (10(-6) M) had no effect on the maximal carbachol-induced contraction in trabecular meshwork strips. Methoctramine induced a significant relaxation at concentrations of 10(-7), 10(-6) and 5 x 10(-6) M even in the presence of m1- and m3-receptor antagonists. CONCLUSION: These data indicate that in addition to the m3-receptor subtype present in the trabecular meshwork this tissue also features the m2 receptor. This receptor is partly involved in the regulation of trabecular meshwork contractility, suggesting that outflow facility might be influenced through this receptor.

Fibroblast growth factor receptor 2 (FGFR2) in brain neurons and retinal pigment epithelial cells act via stimulation of neuroendocrine L-type channels (Ca(v)1.3).

In contrast to the fibroblast growth factor receptor 1 (FGFR1), little is known about intracellular signaling of FGFR2. The signaling cascade of FGFR2 was studied using the perforated patch configuration of the patch-clamp technique in cultured rat retinal pigment epithelial (RPE) cells that express both FGFR1 and FGFR2. Interaction of signaling proteins was studied using immunoprecipitation techniques with membrane proteins from RPE cells and freshly isolated rat brain. When Ba(2+) currents through L-type channels were studied, extracellular application of bFGF (10 ng/ml) led to a shift of the steady-state activation to more negative values. In 50% of cells, an additional increase in maximal current amplitude was observed. This effect was blocked by the tyrosine kinase inhibitor lavendustin A (10(-5) M) but was not influenced by the FGFR1 blocker SU5402 (2 x 10(-5) M) or by the blocker for src-kinase herbimycin A (10(-5) M). Immunoprecipitation of FGFR2 led to coprecipitation of alpha 1D Ca(2+) channel subunits and precipitation of alpha 1D subunits led to coprecipitation of FGFR2. Immunoprecipitation of FGFR1 did not result in the coprecipitation with alpha 1D Ca(2+) channel subunits. The coprecipitation results were comparable when using brain tissue and RPE cells. The alpha 1D subunit-specific band were stained with antiphosphotyrosine antibodies. We conclude that FGFR2 acts via a different signaling cascade than FGFR1. This cascade involves an src-kinase-independent, close functional interaction of FGFR2 and the alpha subunit of neuroendocrine L-type channels.

Mediation of calcium-independent contraction in trabecular meshwork through protein kinase C and rho-A.

PURPOSE: Inhibition of protein kinase C (PKC) and rho-kinase (ROCK) may represent a new way of influencing outflow facility through isolated relaxation of the trabecular meshwork (TM). This work was performed to investigate the existence of calcium-independent contraction in this smooth-muscle-like tissue and its modulation by targeting the rho-guanosine triphosphatase (GTPase)-mediated pathway. METHODS: Isometric tension measurements of bovine TM and ciliary muscle (CM) were performed. Intra- and extracellular calcium buffering was accomplished with EGTA and 1, 2-bis(2-aminophenoxy)-ethane-N,N:,N:,N:',N:'-tetra-acetic acid tetrakis/acetoxymethhyl ester (BAPTA-AM) followed by stimulation of PKC with phorbolester (PMA) or 4alpha-phorbol. Calcium-independent contraction was blocked using the highly specific ROCK inhibitor Y-27632. Western blot analysis and immunoprecipitation was performed using human TM cells. RESULTS: In TM, carbachol induced partial contraction under conditions of extracellular calcium depletion (22. 1% +/- 2.3% versus 100%, n = 9). The membrane-permeable calcium chelator BAPTA-AM completely blocked this response (1.1% +/- 1.4% versus 100%, n = 9). When calcium was completely blocked, PMA induced contraction in TM (16.7% +/- 5.9% versus 100%, n = 9) but not in CM (1.8% +/- 2.5% versus 100%, n = 6). The inactive PMA analogue 4alpha-phorbol did not induce contraction, indicating that activation of PKC is involved in this contractile response. The ROCK inhibitor Y-27632 completely blocked the calcium-independent PMA-induced contraction in TM. Western blot analysis and immunoprecipitation revealed the expression of the rho-A protein in human TM cells. CONCLUSIONS: The data indicate that contrary to CM, the TM features calcium-independent contractile mechanisms linked to rho-A and PKC isoforms that do not require calcium for activation. ROCK inhibitors may allow specific modulation of the TM to enhance outflow facility, thus lowering intraocular pressure.

Activation of neuroendocrine L-type channels (alpha1D subunits) in retinal pigment epithelial cells and brain neurons by pp60(c-src).

The aim of this study is to characterize the subtype of tyrosine kinase-regulated L-type Ca(2+) channels in retinal pigment epithelial (RPE) cells. Ca(2+) channel alpha1D-subunits were enriched by immunoprecipitation from membrane proteins isolated from rat RPE cells. Western blot analysis of the precipitates revealed coprecipitation of pp60(c-src). In addition, in precipitates obtained with antibodies against pp60(c-src), alpha1D-subunits were identified. The same was observed in immunoprecipitations from rat brain neurons. Tyrosine phosphorylation of alpha1D-subunits was confirmed using anti-phosphotyrosine antibodies. Ba(2+) currents through L-type channels in cultured rat RPE cells were increased by intracellular application of active pp60(c-src) (30 U/ml) (heat-inactivated pp60(c-src) had no effect). Thus, L-type channels of the neuroendocrine subtype can be expressed in epithelial cells and are activated by tyrosine kinase of the src subtype. This kind of regulation is also suggested for brain-derived neurons.

Influence of muscarinic agonists and tyrosine kinase inhibitors on L-type Ca(2+)Channels in human and bovine trabecular meshwork cells.

Trabecularmeshwork (TM), a smooth muscle-like tissue with contractile properties, is involved in the regulation of aqueous humor outflow. However, little is known about the regulation of Ca(2+)influx in trabecular meshwork cells. We investigated the influence of acetylcholine and tyrosine kinases on Ca(2+)conductances of bovine TM (BTM) and human TM (HTM) cells using the perforated-patch configuration of the patch-clamp technique and measurements of intracellular free Ca(2+)([Ca(2+)](i)). Depolarization of the cells in the presence of 10 m m Ba(2+)or Ca(2+)led to an activation of inward currents at potentials positive to -30 mV with characteristics typical of L-type Ca(2+)currents: when using 10 m m Ba(2+), maximal inward current and inactivation time constant (tau) increased; the L-type Ca(2+)channel blocker nifedipine (1 microm) reduced and the L-type Ca(2+)channel agonist BayK8644 (5 microm) enhanced maximal inward current. Acetylcholine (100 microm) and carbachol (1 microm) led to an increase in inward Ba(2+)current whereas application of the tyrosine kinase inhibitors genistein (50 microm) and lavendustin A (20 microm) resulted in a decrease in inward current. The application of daidzein (10 microm), an inactive analog of genistein had no effect. Depolarization of the cells with 135 m m K(+)or direct stimulation of L-type channels by application of BayK 8644 led to an increase in [Ca(2+)](i). Carbachol (1 microm) induced an increase in [Ca(2+)](i)which was decreased by application of the tyrosine kinase inhibitor genistein (50 microm). We conclude that HTM and BTM cells express voltage-dependent L-type Ca(2+)channels that influence intracellular Ca(2+)concentration and thus may modulate TM contractility. The activity of L-type Ca(2+)currents is influenced by muscarinic agonists and tyrosine kinases.