[Prospective noninterventional BLUE SKY study evaluating the efficacy of brolucizumab in treatment-naïve and previously treated patients with neovascular AMD]. / Prospektive nichtinterventionelle BLUE SKY-Studie zur Beurteilung der Wirksamkeit von Brolucizumab bei unbehandelten und vorbehandelten Patienten mit neovaskulärer AMD.

Intravitreal injection of anti-vascular endothelial growth factor (VEGF) is the standard treatment for patients with neovascular age-related macular degeneration (nAMD). In addition to the approved substances ranibizumab (Lucentis®, Novartis) and aflibercept (Eylea®, Bayer), bevacizumab (Avastin®, Roche) is also available. Furthermore, brolucizumab (Beovu®, Novartis) has been approved and has been available in Germany since April 2020. The multicenter, noninterventional prospective BLUE SKY study investigates brolucizumab treatment with different schemes in 600 treatment-naive and pretreated nAMD patients in routine clinical practice over a 24-month period. Besides general patient data, visual acuity and treatment data will be documented. Fluorescein angiography, fundus photography, spectral domain optical coherence tomography and swept-source optical coherence tomography angiography will be performed and analyzed by reading centers. The focus of the analysis will be on the intraretinal and subretinal fluid distribution as well as morphological MNV changes and injection frequency. Also, safety and adverse drug effects of brolucizumab, with a specific focus on inflammatory complications, particularly retinal (occlusive) vasculitis will be evaluated.

Comparison of cell-associated and soluble galactosyltransferase isoenzymes from a human bladder transitional cell carcinoma line.

We investigated the asialo-agalactofetuin galactosyltransferase solubilized by Triton X-100 from a human bladder transitional cell carcinoma line (cell-associated form). The specific activity of this enzyme was dependent on cell population density, being about 50% higher in cells from confluent than from sparse cultures. We compared the properties of this enzyme with those of a galactosyltransferase isoenzyme present in the culture medium (soluble form). Electrophoresis on nondenaturing polyacrylamide gels showed the two forms to be isoenzymes, in that the mobility of the soluble enzyme was greater than that of the cell-associated enzyme. The isoenzymes differed in that the Km for UDP-galactose of the cell-associated enzyme (1 X 10(-5) M) was one-half that of the soluble isoenzyme. The isoenzymes differed by 1 order of magnitude in their affinity for a fetuin-derived acceptor with a Km of 16 X 10(-5) M for the cell-associated and 1.2 X 10(-5) M for the soluble form, although the Km for ovalbumin and asialomucin as acceptor was similar for both. Both enzymes were active over a broad pH range and their response to divalent cations was the same: the most efficient cation was Mn2+; but modest activity was detected in the presence of either Cd2+ or Co2+. As determined by gel filtration on Sepharose 6B, the cell-associated galactosyltransferase showed a molecular weight of 66,000, whereas that of the soluble form was 51,000. Limited proteolysis of the cell-associated enzyme with thermolysin and subsequent analysis by nondenaturing polyacrylamide gel electrophoresis demonstrated that the cell-associated enzyme could be converted to an isoenzyme showing the same electrophoretic mobility as the soluble enzyme present in the culture medium, presumably by removal of a portion of the peptide chain. The same result was obtained by treating the cell-associated enzyme with a cell extract. This suggests but does not prove that the soluble enzyme secreted or shed into the medium is produced from the cell-associated form by an endogenous protease.

Conversion of the amphiphilic galactosyltransferase from human mammary carcinoma cells to an active hydrophilic enzyme form by limited proteolysis.

As analyzed by a phase-separation technique, the Triton X-114 extract of human mammary carcinoma cells (MCF-7 cells) contain an amphiphilic form of galactosyltransferase (UDPgalactose: D-glucose 4-beta-D-galactosyltransferase, EC 2.4.1.22), while the galactosyltransferase activity released by these cells represents a hydrophilic form of the enzyme. When the amphiphilic galactosyltransferase was subjected to limited proteolysis with thermolysin, this treatment generated a hydrophilic form of the enzyme. With respect to Km for UDPgalactose the kinetic data were very similar for the amphiphilic, for the released and the hydrophilic galactosyltransferases produced by proteinase treatment. Differences were detected in electrophoretic and gel chromatographic properties. The hydrophilic enzymes showed a greater electrophoretic mobility on non-denaturing polyacrylamide gels than did the amphiphilic form. On Sepharose 6B column chromatography, the amphiphilic galactosyltransferase appeared to be of higher molecular weight than the hydrophilic enzyme.

The effect of retinoids on the activity of the membrane form of galactosyltransferase, studied in an enzyme/liposome model system.

In the present study the effect of retinoids on the membrane form of galactosyltransferase was tested. A model system consisting of pure bovine milk galactosyltransferase and phosphatidylserine vesicles was used for this investigation. Retinol, retinal and retinylphosphate were able to overcome the modulating effect of phosphatidylserine, that is, activated the enzyme. Retinoic acid and retinylpalmitate were ineffective in this system.

Beta-oxidation in peroxisomes of brown adipose tissue.

Peroxisomes and mitochondria from brown adipose tissue of the rat were separated by differential pelleting and isopycnic gradient centrifugation. Both fractions oxidized palmitoyl-CoA with comparable specific activities. Unlike the mitochondrial beta-oxidation the peroxisomal activity was not influenced by carbon monoxide. Peroxisomal beta-oxidation together with carnitine acetyl-transferase, which is also located in peroxisomes, might be involved in chemical thermogenesis by delivering acetyl groups to the mitochondria.

Semicarbazide-sensitive amine oxidase catalyzes endothelial cell-mediated low density lipoprotein oxidation.

OBJECTIVE: Deamination products of semicarbazide-sensitive amine oxidases (SSAO), i.e. aldehydes, superoxide and ammonia have been shown to initiate vascular damage. SSAOs are copper-enzymes, present in endothelial (EC), smooth muscle cells (SMC) and in blood. Transition metals ions (Cu, Fe) mediate the oxidative (atherogenic) modification of LDL by SMC and EC. The physiological source of the active metal ions is still under debate. We hypothesize that SSAOs may catalyze LDL oxidation by endothelial cells via enzyme-complexed Cu++. METHODS: EC isolated from human umbilical veins and cultured in 35 mm wells in RPMI-1640 medium were used as LDL oxidation system. RESULTS: Diamine oxidase (DAO), a SSAO which activity is elevated in tissues and sera of diabetic patients, catalyzes the oxidation of LDL by EC. In the presence of purified DAO (0.07 to 70 U/l) LDL oxidation was increased up to 10-fold as measured by thiobarbituric acid reactive substance (TBARS) formation as well as apoprotein modification of LDL. Chemical blockage of the SSAO substrate binding site did not inhibit the catalytic effect of DAO on LDL oxidation. Denaturation of the enzyme did not destroy the ability of the preparation to facilitate LDL oxidation by EC. The potential of the enzyme to catalyze LDL oxidation was not suppressed in the presence of serum. However, selective removing of enzyme-copper completely abolished the ability of the enzyme to trigger cell-mediated LDL oxidation. CONCLUSION: DAO, beside generating angiopathic deamination products, has the potential to act as a pathophysiological catalyst of LDL atherogenic modification by vascular cells.

Phosphorylation of tyrosine prevents dityrosine formation in vitro.

Treatment of L-tyrosine in a peroxidase/H2O2 system results in the formation of dityrosine. However, the phosphoester derivative of tyrosine, O-phospho-L-tyrosine, was unable to form dityrosine even in mixtures with free L-tyrosine. Dephosphorylation of O-phospho-L-tyrosine by alkaline phosphatase followed by horseradish peroxidase/H2O2 treatment resulted in the formation of dityrosine. Our in vitro results indicate that phosphorylation/dephosphorylation of L-tyrosine may regulate dityrosine formation, and is supposed to play an important role in protein-protein interactions, i.e. cross-linking.

Tyrosine phosphorylation blocks tyrosine free radical formation and, hence, the hormonogenic iodination reaction.

It is known that a tyrosine free radical is produced during the hormonogenic iodination reaction of tyrosine residues on thyroglobulin. In the hormonogenic region of thyroglobulin, phosphorylated tyrosine residues have been detected. Using an vitro tyrosine iodinating system we report that the hormonogenic reaction cannot go off if tyrosine becomes phosphorylated. Enzymatic dephosphorylation of the modified amino acid restored the ability of the molecule to become iodinated. Considering the mechanism of the tyrosine free radical formation, these observations are due to the inability of the phosphorylated amino acid to form a free radical. Our data may suggest a putative regulatory mechanism in thyroid hormone synthesis by phosphorylation of hormonogenic tyrosine residues on thyroglobulin.

Tyrosine: an inhibitor of LDL oxidation and endothelial cell cytotoxicity initiated by superoxide/nitric oxide radicals.

Tyrosyl radicals can catalyze LDL oxidation. In addition to their LDL oxidizing ability, superoxide (O2.-)/nitric oxide (NO.) generate phenoxyl radicals when reacting with tyrosine. Therefore we tested if tyrosine can act as a pro-oxidant in O2.-/NO.-initiated LDL oxidation. When LDL was exposed to O2.-/NO., tyrosine exerted a strong inhibitory effect on O2.-/ NO.-initiated LDL oxidation as measured by TBARS formation and alteration in electrophoretic mobility of LDL. Tyrosine was also able to protect human endothelial cells from the cytotoxic effect of O2.-/NO.. Because O2.-/NO. can occur in vivo, the results may indicate that serum-free tyrosine could act as an efficacious physiological antioxidant in case of O2.-/NO.-initiated LDL oxidation and endothelial cell cytotoxicity.

Salicylate promotes myeloperoxidase-initiated LDL oxidation: antagonization by its metabolite gentisic acid.

The oxidative modification of low density lipoprotein (LDL) may play a significant role in atherogenesis. Tyrosyl radicals generated by myeloperoxidase (MPO) can act as prooxidants of LDL oxidation. Taking into consideration, that monophenolic compounds are able to form phenoxyl radicals in presence of peroxidases, we have tested salicylate, in its ability to act as a prooxidant in the MPO system. Measurement of conjugated dienes and lipid hydroperoxides were taken as indicators of lipid oxidation. Exposure of LDL preparations to MPO in presence of salicylate revealed that the drug could act as a catalyst of lipid oxidation in LDL. The radical scavenger ascorbic acid as well as heme poisons (cyanide, azide) and catalase were inhibitory. The main metabolite of salicylic acid, gentisic acid, showed inhibitory action in the MPO system. Even when lipid oxidation was maximally stimulated by salicylate the LDL oxidation was efficaciously counteracted in presence of gentisic acid at salicylate/gentisic acid ratios that could be reached in plasma of patients receiving aspirin medication. Gentisic acid was also able to impair the tyrosyl radical catalyzed LDL peroxidation. The results suggest that salicylate could act like tyrosine via a phenoxyl radical as a catalyst of LDL oxidative modification by MPO. But the prooxidant activity of this radical species is effectively counteracted by the salicylate metabolite gentisic acid.

Salicylate inhibits LDL oxidation initiated by superoxide/nitric oxide radicals.

Simultaneously produced superoxide/nitric oxide radicals (O2*-/NO*) could form peroxynitrite (OONO-) which has been found to cause atherogenic, i.e. oxidative modification of LDL. Aromatic hydroxylation and nitration of the aspirin metabolite salicylate by OONO- has been reported. Therefore we tested if salicylate may be able to protect LDL from oxidation by O2*-/NO* by scavenging the OONO reactive decomposition products. When LDL was exposed to simultaneously produced O2*-/NO* using the sydnonimine SIN-1, salicylate exerted an inhibitory effect on LDL oxidation as measured by TBARS and lipid hydroperoxide formation and alteration in electrophoretic mobility of LDL. The cytotoxic effect of SIN-1 pre-oxidised LDL to endothelial cells was also diminished when salicylate was present during SIN-1 treatment of LDL. Spectrophotometric analysis revealed that salicylate was converted to dihydroxybenzoic acid (DHBA) derivatives in the presence of SIN-1. 2,3- and 2,5-DHBA were even more effective to protect LDL from oxidation by O2*-/NO*. Because O2*-/NO* can occur in vivo, the results may indicate that salicylate could act as an efficacious inhibitor of O2*-/NO* initiated atherogenic LDL modification, thus further supporting the rationale of aspirin medication regarding cardiovascular diseases.

Homocysteine promotes the LDL oxidase activity of ceruloplasmin.

Ceruloplasmin (CP) oxidises low density lipoprotein (LDL). The oxidising potential depends on the formation of Cu(+)-CP which is redox-cycled during oxidation. Homocysteine (HCY) reduces free Cu(2+), potentiating its cell-damaging property. We show that HCY enhanced LDL oxidation by CP, but did not activate the LDL oxidising potential of Cu(2+)-diamine oxidase. Selective removal of the redox-active Cu(2+) abolished the LDL oxidase activity of CP. However, HCY partially restored the LDL oxidase activity of redox-copper depleted CP, indicating that the remaining six copper atoms in CP may also be involved in the process. Spectroscopic and oxidation inhibition studies using the Cu(+)-reagent bathocuproine revealed that HCY induced Cu(+)-CP formation, thus promoting its LDL oxidase activity.

p-Hydroxyphenylacetaldehyde, the major product of tyrosine oxidation by the activated myeloperoxidase system can act as an antioxidant in LDL.

The oxidative modification of low density lipoprotein (LDL) may play a significant role in atherogenesis. HOCl generated by the myeloperoxidase/H2O2/Cl- system of activated neutrophils may be operative in vivo making LDL atherogenic. Tyrosine has been found to be oxidized by HOCl to p-hydroxyphenylacetaldehyde (p-HA) capable of modifying phospholipid amino groups in LDL. As an amphiphatic phenolic compound, p-HA may have the potential to act as an antioxidant in the lipid phase of LDL. The present results show that (a) tyrosine exerts a protective effect on LDL modification by HOCl, (b) p-HA could act as antioxidant associated with the lipoprotein preventing cell- and transition metal ion-mediated LDL oxidation and (c) p-HA was able to scavenge free radicals.

The salicylate metabolite gentisic acid, but not the parent drug, inhibits glucose autoxidation-mediated atherogenic modification of low density lipoprotein.

Oxidation of low density lipoprotein (LDL) by glucose-derived radicals may play a role in the aetiology of atherosclerosis in diabetes. Salicylate was shown to scavenge certain radicals. In the present study, aspirin, salicylate and its metabolites 2,5- and 2, 3-dihydroxybenzoic acid (DHBA) were tested for their ability to impair LDL oxidation by glucose. Only the DHBA derivatives, when present during LDL modification, inhibited LDL oxidation and the increase in endothelial tissue factor synthesis induced by glucose oxidised LDL. The LDL glycation reaction was not affected by DHBA. The antioxidative action of DHBA may be attributed to free radical scavenging and/or chelation of transition metal ions catalysing glucose autoxidation.

Oral contraceptives that contain ethinyl estradiol (0.035 mg) and cyproterone acetate (2 mg) inhibit leukocyte transmigration through endothelial cell monolayers.

OBJECTIVE: To investigate the influence of ethinyl estradiol and cyproterone acetate in oral contraceptives on leukocyte migration through endothelial cell monolayers. DESIGN: Experimental in vitro prospective study. SETTING: An academic research laboratory. INTERVENTION(S): Endothelial cells were cultured on microporous membranes to produce monolayers. Polymorphonuclear leukocytes were used in a previously described migration assay (n = 7). The amount of untreated polymorphonuclear leukocytes that migrated through untreated endothelial cell monolayers was used as a control and set at 100%. In addition, a leukocyte adhesion assay was used. MAIN OUTCOME MEASURE(S): Leukocyte adhesion to and transmigration through endothelial cell monolayers. RESULT(S): Ethinyl estradiol and cyproterone acetate inhibited the migration of polymorphonuclear leukocytes through endothelial cell monolayers significantly (67% +/- 6.4%) when both cell types were treated to simulate in vivo conditions. The adhesion assay produced similar results. CONCLUSION(S): Ethinyl estradiol and cyproterone acetate were identified as potent inhibitors of leukocyte migration through endothelial cell monolayers.

Genistein prevents the glucose autoxidation mediated atherogenic modification of low density lipoprotein.

Hyperglycemia has been assumed to be responsible for oxidative stress in diabetes. In this respect, glucose autoxidation and advanced glycation end products (AGE) may play a causal role in the etiology of diabetic complications as e.g. atherosclerosis. There is now growing evidence that the oxidative modification of LDL plays a potential role in atherogenesis. Glucose derived oxidants have been shown to peroxidise LDL. In the present study, genistein, a compound derived from soy with a flavonoid chemical structure (4', 5, 7-trihydroxyisoflavone) has been evaluated for its ability to act as an antioxidant against the atherogenic modification of LDL by glucose autoxidation radical products. Daidzein, (4',7-dihydroxyisoflavone) an other phytoestrogen of soy, was tested in parallel. Genistein--in contrast to daidzein--effectively prevented the glucose mediated LDL oxidation as measured by thiobarbituric acid-reactive substance formation (TBARS), alteration in electrophoretic mobility, lipid hydroperoxides and fluorescence quenching of tryptophan residues of the lipoprotein. In addition the potential of glucose-oxidized LDL to increase tissue factor (TF) synthesis human endothelial cells (HUVEC) was completely inhibited when genistein was present during LDL oxidative modification by glucose. Both phytoestrogens did not influence the nonenzymatic protein glycation reaction as measured by the in vitro formation of glycated LDL. As the protective effect of genistein on LDL atherogenic modification was found at glucose/genistein molar ratios which may occur in vivo, our findings support the suggested beneficial action of a soy diet in preventing chronic vascular diseases and early atherogenic events.

Dexamethasone inhibits leukocyte migration through endothelial cells towards smooth muscle cells.

Leukocyte interactions with endothelial cell monolayers (ECM) and smooth muscle cells (SMC) play an important role during inflammatory processes. Several studies describe an inhibitory effect of dexamethasone on polymorphonuclear leukocytes (PMNL), endothelial cell function, and interleukin-1 (IL-1) release. Aim of the current study was to investigate the influence of dexamethasone on leukocyte migration through an endothelial cell monolayer towards SMC-layers stimulated by tumor necrosis factor-alpha (TNF-alpha). Using a recently developed triple chamber migration system, SMC-layers were cultured on the bottom of a 24-well plate. On the upper surface of the first filter, ECM were cultured, the second filter was a collecting filter. The amount of leukocyte migration through ECM towards TNF-alpha-stimulated smooth muscle cell layers with and without dexamethasone-pretreatment was measured using a fluorescence technique. The pretreatment of SMC-layers with dexamethasone reduced the amount of leukocyte migration down to 92 +/- 8.8% (0.001 mM, p=n.s.), to 67 +/- 5.7% (0.01 mM, p<0.05), to 53 +/- 4.6% (0.1 mM, p<0.05), and to 41 +/- 5.0% (1 mM, p<0.05). In conclusion, dexamethasone treatment of smooth muscle cell layers inhibits leukocyte migration through ECM towards smooth muscle cell layers. The inhibition seems to be due to a decrease in IL-1 release. Treatment of all cell types, PMNL, endothelial cells, as well as smooth muscle cell layers, simulating an in-vivo situation, seems to have an additive effect.

Rapid, fluorescence-based assay for microtiter plates to test drug influences on neutrophil transmigration through endothelial cell monolayers.

Investigation of drug interactions between blood cells and endothelium is of high interest. The current study describes the development of a rapid fluorescence-based leukocyte transmigration system through endothelial cell monolayers for investigation of drug influences. To test the new assay, endothelial cells were cultured on microporous filters, pore size 3.0 microm, in 96-well-plates. Freshly isolated neutrophils were seeded on endothelial cell monolayers and transmigrated cells were measured after incubation for three hours. Migration of non-stimulated neutrophils through non-stimulated endothelial cell monolayer was used as control and set as 100%. The influence of the non-steroidal anti-inflammatory drug diclofenac was investigated. Assay precision tests were done using intraassay (within-day variability) and interassay (day-to-day variability) controls. Transmigration rate was decreased to 53 +/- 6.8% SD (diclofenac 0.7 microg/mL). Different concentrations showed a dose dependent effect (0.07 microg/mL: 97 +/- 9.5%, 7 microg/mL: 37 +/- 4.7%). Analysis of assay accuracy of the new 96-well-sized transmigration assay showed reliable results (coefficient of variation: intraassay 8.2 %; interassay 11.8%). In conclusion, this new, rapid, and sample saving 96-well-microtiter transmigration assay allows examination of drug influence on neutrophil migration through endothelial cell monolayers. Moreover, this assay can also be used for other cell-cell interactions.

Modulation of biological tyrosine reactions by tyrosine phosphorylation.

This review focuses on the influence of tyrosine phosphorylation on the biological reactions of tyrosine. Reactions that are modulated by this amino acid modification include dityrosine formation, thyroid hormone synthesis, and DOPA formation. In addition, we show that the reactivity of tyrosine in the common Lowry method of determination of protein concentrations is lost upon phosphorylation of the amino acid.

"Wiener klinische Wochenschrift": publication patterns 1990-2000.

The impact factor (Institute for Scientific Information, ISI, Philadelphia, PA, USA) is a widespread used and acknowledged source for judging the quality of a researcher. In addition the "Science Citation Index (SCI)" [Institute for Scientific Information (ISI)] provides the scientific community with a citations database indicating the number of cited references in indexed articles. For several reasons, the SCI seems to be more relevant. To evaluate the quality of the journal "Wiener klinische Wochenschrift", we assessed how often contributors to this journal have been cited during the last decade and which contributions have ranked as top papers. Moreover, with the aim of a more objective type of scientific evaluation, we have employed a new score, the "Citation Per Time Score (CPT-Score--the SCI divided by the number of years of observation, starting one year after publication). We have evaluated the total SCI for the years 1990 to 1994 and for the years 1995 to 1999. The number of total citations between the appearance of an article and the year 2000 was analyzed. The highest ranked publication of the whole decade (1990-2000) was a paper by G. Stanek with 40 citations. Furthermore, we have evaluated the "Top Ten Papers" of the journal. Interestingly, a steady increase of the total citation index of the journal "Wiener klinische Wochenschrift" over the last decade could be demonstrated. This study clearly shows that the citation rate of an article is not determined by the impact factor of the journal but rather the quality of the contribution. Moreover, the citation analysis of papers published in the last ten years in the "Wiener klinische Wochenschrift" shows a satisfactory citation rate for articles published in this journal. It is therefore definitely attractive to submit an article to a journal such as the "Wiener klinische Wochenschrift".