[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.

[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.

[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.

[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.

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.

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.

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.

Role of protein tyrosine kinase on regulation of trabecular meshwork and ciliary muscle contractility.

PURPOSE: Trabecular meshwork and ciliary muscle express properties of smooth muscle cells. The contractility of trabecular meshwork and ciliary muscle is differently modulated by various agents. To reveal contractile regulatory processes, the effects of activation and inhibition of protein tyrosine kinases (PTKs) and their interaction with other protein kinases on contractility were measured. METHODS: Measurements of isometric tension were performed on isolated bovine trabecular meshwork and ciliary muscle strips using a custom-built, electromagnetic, force-length transducer. Protein tyrosine kinase (PTK) was stimulated by epidermal growth factor (EGF) and was inhibited by genistein or tyrphostin 51. Protein kinase C (PKC) was inhibited by chelerythrine or NPC-15437 and protein kinases A and G (PKA-PKG) by H8. RESULTS: Isolated strips were precontracted by applying carbachol 10(-6) M for 30 minutes (100% carbachol maximum contraction). Inhibition of PTK evoked a maximum relaxation of 79.2+/-4.2% in trabecular meshwork and of 38.1+/-3.1% in ciliary muscle (n=8). Inhibition of PKC or PKA-PKG induced relaxations only in trabecular meshwork. When PTK and PKC or PKA-PKG were inhibited, the relaxation induced by inhibition of PTK was additive to inhibition of the other protein kinases. Stimulation of a receptor with PTK activity by EGF induced a relaxation in trabecular meshwork and a contraction in ciliary muscle precontracted by carbachol. When trabecular meshwork and ciliary muscle were activated by EGF, inhibition of PTK by genistein relaxed the cell preparations. CONCLUSIONS: Inhibition of PTK induces more prominent relaxation in trabecular meshwork than in ciliary muscle. The effects of inhibition of PTK on relaxation are independent of inhibition of PKC and PKA-PKG. The signaling cascade after activation of a tyrosine kinase receptor by EGF is differently modulated in trabecular meshwork and ciliary muscle. The effect of genistein on relaxation is probably not directly related to the EGF receptor. PTK inhibitors are possible agents for the development of novel antiglaucoma drugs.

Characterization of maxi-K-channels in bovine trabecular meshwork and their activation by cyclic guanosine monophosphate.

PURPOSE: Electrophysiological characterization of trabecular meshwork cells and investigation of their response to elevation of cytosolic cyclic guanosine monophosphate (cGMP). METHODS: Bovine trabecular meshwork cells were cultured according to established methods and were studied, using the whole-cell and single-channel configurations of the patch-clamp technique. RESULTS: In single-channel experiments, cells expressed a channel with characteristics typical of maxi-K-channels. The channel was densely distributed in the membrane and had a high conductance of 326 +/- 4 pS (Pico Siemens) (symmetrical 150 mmol/l KCl; 37 degrees C) for potassium and negligible conductance for sodium (0.9 +/- 1 pS). The open probability could be elevated by depolarization, increasing cytosolic calcium, or adding adenosine triphosphate (1 mmol/ l). The channel could be blocked by external charybdotoxin (10(-8) mol/1), external TEA+ tetraethyl ammonium chloride (1 mmol/l) and by internal Ba2+ (10 mmol/l), whereas external Ba2+ and internal TEA+ (10 mmol/l) had no effect. In whole-cell experiments, trabecular meshwork cells displayed a strong outward conductance. Part of this conductance (35 +/- 5%) could be blocked by charybdotoxin and stimulated by ionomycin (10(-5) mol/1). Addition of 8-bromo-cGMP (10(-3) mol/1) stimulated the current to 290 +/- 57% (n = 4) of the original level, charybdotoxin led to a reduction of this current to 156 +/- 28% of the initial value. CONCLUSIONS: Trabecular meshwork cells express maxi-K-channels. These channels can be stimulated by raising internal cGMP levels and are known for their importance in smooth muscle relaxation. The results in this study supply further evidence that trabecular meshwork displays smooth muscle-like properties and contributes to the clarification of the mechanism leading to the relaxation of trabecular meshwork by nitrate and nonnitrate vasodilatators.

Stimulation of maxi-K channels in trabecular meshwork by tyrosine kinase inhibitors.

PURPOSE: Muscarinic agonists contract and tyrosine kinase inhibitors relax precontracted trabecular meshwork, a smooth muscle-like tissue involved in the regulation of aqueous humor outflow. The effect of tyrosine kinase inhibitors on membrane currents of cells stimulated by acetylcholine was examined. METHODS: Cells from bovine trabecular meshwork were studied using both the perforated patch-clamp technique with nystatin and the single-channel technique. RESULTS: Application of the tyrosine kinase inhibitor genistein (5 x 10(-5) M) on trabecular meshwork cells stimulated with acetylcholine resulted in a reversible increase in outward current to 578%+/-154% (n = 16) of the initial current level. The effect of genistein was dose dependent. Reversal potential was hyperpolarized by 15+/-3 mV (n = 9). Tyrphostin 51, a synthetic inhibitor of tyrosine kinases, had the same effect (433%+/-46%; n = 7). Daidzein, a nonactive structural analogue of genistein, had no effect (n = 4). The stimulation of outward current by tyrosine kinase inhibitors was blocked by substitution of tetraethylammonium (TEA+) for potassium, whereas the potassium channel blockers glibenclamide (K-ATP) and apamin (low-conductance calcium-activated potassium channel) had no effect. Blockage of the high-conductance calcium-activated potassium channel (maxi-K) by charybdotoxin or iberiotoxin (10(7) M) suppressed 86%+/-18% (n = 4) of the response. Depleting the cells of calcium did not have an effect on the current stimulated by genistein. In the excised inside-out configuration, open probability increased to 417%+/-39% (n = 3) after exposure to genistein. CONCLUSIONS: In trabecular meshwork, tyrosine kinase inhibitors activate maxi-K (K(Ca)) channels. Hyperpolarization caused by efflux of potassium could lead to the relaxation of trabecular meshwork by tyrosine kinase inhibitors.

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.

The effects of protein kinase C on trabecular meshwork and ciliary muscle contractility.

PURPOSE: The possible role of protein kinase C (PKC) inhibitors in novel pressure-lowering drugs is currently under investigation. To gain further insight into regulation of contractility by PKC in trabecular meshwork (TM) and ciliary muscle (CM), the effects of various PKC inhibitors and activators were tested. METHODS: Isometric tension measurements of bovine TM and CM strips were performed. PKC was stimulated by phorbol ester and by the diacylglycerol analogue diC8. PKC blockade was accomplished using H7 and myristoilated PKC substrate (mPKC). Western blot analysis was used to identify specific PKC isoforms in human trabecular meshwork (HTM), human ciliary muscle (HCM), and bovine TM and CM. RESULTS: In tissues precontracted by carbachol PKC antagonist H7 led to a relaxation of TM (25+/-7.2 versus 100%; n = 8) with no effect on CM. mPKC substrate selectively blocks PKC. This substance led to relaxation of TM (32.8+/-7.4 versus 100%, n = 7), whereas CM was not affected. PMA at concentrations of 10(-6) M led to a slow contraction of both tissues that was more marked in TM. DiC8 and 4alpha-phorbol had no effect on contractility. Western blot analysis revealed expression of calcium-dependent PKC-alpha and calcium-independent PKC-epsilon isoforms in HTM and HCM. PKC-epsilon expression was more pronounced in HTM than in HCM. Similar PKC isoform expression was found in native bovine tissue. CONCLUSIONS: PKC isoforms show different tissue distributions in human and bovine TM and CM. Contractility differences exist in both tissues in response to PKC antagonists and agonists. The data indicate that PKC may be involved in regulation of aqueous humor outflow by the TM. Thus, inhibition of PKC may represent a new way of influencing outflow facility through isolated relaxation of TM.

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.

Incomplete belts of tight junctions in cultured non-pigmented human ciliary epithelial cells.

Tight junctions of cultured human non-pigmented ciliary epithelial cells were studied with the freeze-fracture technique and related to the transepithelial electrical resistance of these monolayers. Isolated tight junctional fibrils or small groups and networks of tight junctions sometimes associated with gap junctions were revealed in freeze-fracture images of the lateral plasma membrane. The tight junctions always formed incomplete belts, so that the apical and basolateral plasma membrane domains often were in continuity without morphological evidence of interposed intercellular junctions. The monolayers revealed a transepithelial resistance of 19.7 +/- 2.1 omega.cm2. Protamine induced a reversible increase of the transepithelial resistance of the cultures by 91 +/- 12%, but still the tight junctions formed incomplete belts. We conclude that contrary to complete networks of tight junctions in native non-pigmented ciliary epithelium, cultured monolayers only express incomplete belts of tight junctions which may be the morphological correlate of the relatively low transepithelial resistance of these monolayers. Interpretations on transepithelial transport and permeability characteristics of these cultures have to take into account the differences in junctional morphology from their native epithelium.

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.

[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.