Serum vascular cell adhesion molecule-1 predicts significant liver fibrosis in non-alcoholic fatty liver disease.

BACKGROUND: Non-alcoholic fatty liver disease (NAFLD) is the most common chronic liver disease worldwide and is strongly associated with obesity, dyslipidemia and insulin resistance. NAFLD often presents as simple steatosis (NAFL) but can progress to non-alcoholic steatohepatitis (NASH) and fibrosis. Current non-invasive biomarkers are not tailored to identify significant (⩾F2) fibrosis, although recent guidelines recommend a stringent follow-up of this patient population. We and others have reported on the role of pathological angiogenesis in the pathogenesis of NAFLD, highlighting pro-angiogenic factors as potential diagnostic markers. OBJECTIVE: To investigate the applicability of angiogenic and endothelial dysfunction markers as non-invasive diagnostic tools for NASH or NASH-associated fibrosis in obese patients. METHODS: In a prospective cross-sectional study, male patients undergoing bariatric surgery (n=61) and control patients (n=35) were recruited. Serum protein levels and visceral adipose tissue gene expression of endothelial dysfunction and angiogenic markers were analyzed by multiplex bead-based assay and quantitative RT-PCR, respectively. For validation, we recruited a second cohort of patients undergoing bariatric surgery (n=40) and a cohort of NAFLD patients from our outpatient clinic (n=30). RESULTS: We identified serum vascular cell adhesion molecule-1 (VCAM-1) as an independent predictor for ⩾F2 fibrosis (median 14.0 vs 8.7 ng ml-1 in patients with and without significant fibrosis; P<0.0001) with an area under the receiver-operating characteristics (AUROC) curve of 0.80. The cutoff point of 13.2 ng ml-1 showed a sensitivity of 80% and specificity of 83%. In line with these results, VCAM-1 visceral adipose tissue gene expression was also elevated in patients with fibrosis (P=0.030). In the bariatric surgery and clinical validation cohorts, VCAM-1 displayed similar AUROCs of 0.89 and 0.85, respectively. CONCLUSIONS: VCAM-1 levels are able to accurately predict significant (⩾F2) fibrosis in NAFLD patients.

Physical and sensory characterizations of oral coatings of oil/water emulsions.

The physical and sensory properties of oil coatings on the tongue formed by five oil/water emulsions varying in oil content were investigated. A total of 20 subjects processed orally each emulsion for 30 s in triplicate. In vivo fluorescence measurements at the front and back of the anterior tongue were made to quantify the oil fraction deposited at different time points. Calibration lines relating fluorescence intensity to oil fraction were determined using pig tongues at 37.5 °C to mimic oral conditions. The oil fraction on the tongue increased linearly with an increasing oil content of the emulsions. The oil fraction deposited at the back of the anterior tongue was 1.5-2.0× larger than at the front. The intensity of sensory attributes describing after-feel perception was related to the oil fraction by Weber-Fechner's law. This study uses in vivo fluorescence to study food behavior in the mouth and unravel new insights in after-feel perception of emulsions.

Pharmacokinetics of rifampin and clarithromycin in patients treated for Mycobacterium ulcerans infection.

In a randomized controlled trial in Ghana, treatment of Mycobacterium ulcerans infection with streptomycin (SM)-rifampin (RIF) for 8 weeks was compared with treatment with SM-RIF for 4 weeks followed by treatment with RIF-clarithromycin (CLA) for 4 weeks. The extent of the interaction of RIF and CLA combined on the pharmacokinetics of the two compounds is unknown in this population and was therefore studied in a subset of patients. Patients received CLA at a dose of 7.5 mg/kg of body weight once daily, rounded to the nearest 125 mg. RIF was administered at a dose of 10 mg/kg, rounded to the nearest 150 mg. SM was given at a dose of 15 mg/kg once daily as an intramuscular injection. Plasma samples were drawn at steady state and analyzed by liquid chromatography-tandem mass spectroscopy. Pharmacokinetic parameters were calculated with the MW/Pharm (version 3.60) program. Comedication with CLA resulted in a 60% statistically nonsignificant increase in the area under the plasma concentration-time curve (AUC) for RIF of 25.8 mg x h/liter (interquartile ratio [IQR], 21.7 to 31.5 mg x h/liter), whereas the AUC of RIF was 15.2 mg x h/liter (IQR, 15.0 to 17.5 mg x h/liter) in patients comedicated with SM (P = 0.09). The median AUCs of CLA and 14-hydroxyclarithromycin (14OH-CLA) were 2.9 mg x h/liter (IQR, 1.5 to 3.8 mg x h/liter) and 8.0 mg x h/liter (IQR, 6.7 to 8.6 mg x h/liter), respectively. The median concentration of CLA was above the MIC of M. ulcerans, but that of 14OH-CLA was not. In further clinical studies, a dose of CLA of 7.5 mg/kg twice daily should be used (or with an extended-release formulation, 15 mg/kg should be used) to ensure higher levels of exposure to CLA and an increase in the time above the MIC compared to those achieved with the currently used dose of 7.5 mg/kg once daily.

The relaxed confocal scanning laser ophthalmoscope.

The development of the Scanning Laser Ophthalmoscope is reviewed from a historical perspective. Since a flying-spot scanning principle for an electro-optical ophthalmoscope was first disclosed in 1950, enabling milestones have included the introduction of the laser and inversion of the usual Gullstrand's configuration of optical pupils in 1977, and the application of the optical principle of confocality by means of double or de-scanning in 1983. As a result, high resolution and high contrast confocal infra-red ophthalmoscopy with a 790 nm diode laser, at video rates, is a major novel imaging modality when compared to traditional optical techniques. This imaging mode is ideal to provide the necessary fiducial landmarks for microperimetry, therapeutic laser and SD-OCT based optical sectioning of the retina. DPSS or He-Ne lasers emitting at 532, 543, 561 or 575 nm are used for complimentary red-free fundus imaging. The diode 790 nm and DPSS 490 nm lasers are also used for fluorescence excitation.

Microperimetry.

In SLO microperimetry a multi-channel overlay graphics frame grabber synchronizes the scanning of an infra-red laser with the modulation of a visible laser source. Graphics are created in the laser raster with a fast 25 ns sub-pixel rise-time 80 MHz acousto-optic modulator, Bragg angle optimized for 532 nm, 543.5 nm, 632.8 nm or 660 nm wavelengths over a 40 dB attenuation range. Our software kernel comprises 4 alternative forced choice (4AFC), parameter estimation by sequential testing (PEST) and manual or automated tracking algorithms based on two-dimensional normalized gray-scale correlation. They enable fast and accurate registration of fixation patterns, precise measurements of potential acuities and retinal sensitivity using a range of background illumination levels. Opto-electronic characteristics of physiologic significance are discussed. Clinical examples are given.

Eligibility criteria for Nd-YAG laser treatment of highly symptomatic vitreous floaters.

Ten eyes of nine patients were treated for very disturbing vitreous floaters with the technique of Nd-YAG laser vitreolysis. The Scanning Laser Ophthalmoscope (SLO) was used to objectivate the position, the size and the motility of the vitreous floaters with respect to the patient's visual axis, which can be precisely located with the SLO. With this technique it was possible to define more precisely some eligibility criteria for Nd-YAG laser treatment of vitreous floaters and to classify the vitreous floaters in ill-suspended and well-suspended floaters in the vitreous body, the well-suspended floaters responding better to treatment compared to the ill-suspended vitreous floaters. The treatment was performed using the Q-Switched Nd-YAG Laser type Nanolas 15S of Alcon.

Chloroperoxidase-catalyzed enantioselective oxidations in hydrophobic organic media.

Chloroperoxidase from Caldariomyces fumago, a peroxidase that performs P450-like chemistry, was immobilized via covalent attachment into polyurethane foam as well as conjugated with a surfactant or polymer via colyophilization. The resulting preparations catalyzed enantio- and regioselective oxidations in hydrophobic organic media with tert-butyl hydroperoxide as the oxidant. Dried PUR-foam immobilized CPO mediated the selective oxidation of indole to 2-oxindole (regioselectivity: 99%) in water-saturated isooctane or 1-octanol. Thioanisole was converted into the corresponding (R)-sulfoxide (ee > 99%) in isooctane medium. The complexes of CPO with sodium octadecylsulphate or ethyl cellulose mediated the oxidation of thioanisole in water-immiscible organic media with variable enantioselectivity due to radical side-reactions. In the presence of alpha-tocopherol, acting as radical scavenger, the (R)-sulfoxide was formed with ee > 90%. The effect of the water activity on the catalytic activity of the complexes was investigated. The CPO complexes likewise mediated the regioselective oxidation of indole into 2-oxindole in water-saturated isooctane or 1-octanol and its kinetics were investigated. The reaction suffered from substrate inhibition when carried out in isooctane.

On the structure of kappa/iota-hybrid carrageenans.

The coil-to-helix transition and temperature dependence of the viscosity of commercial kappa/iota-hybrid carrageenans produced by the red algae Sarcothalia crispata, Mazaella laminarioides, and Chondrus crispus were studied using rheometry and optical rotation. The structure of these kappa/iota-hybrid carrageenans was determined by 1H and 13C NMR spectroscopy combined with monosaccharide composition analysis. The coil-to-helix transitions, measured by polarimetry and rheometry, of the kappa/iota-hybrid carrageenans are significantly different from those of pure kappa- and iota-carrageenan, and from hand-made mixtures thereof. This provides evidence that the kappa/iota-hybrid carrageenans are mixed chains, containing both kappa- and iota-repeating units.

Expression of the Caldariomyces fumago chloroperoxidase in Aspergillus niger and characterization of the recombinant enzyme.

The Caldariomyces fumago chloroperoxidase was successfully expressed in Aspergillus niger. The recombinant enzyme was produced in the culture medium as an active protein and could be purified by a three-step purification procedure. The catalytic behavior of recombinant chloroperoxidase (rCPO) was studied and compared with that of native CPO. The specific chlorination activity (47 units/nmol) of rCPO and its pH optimum (pH 2.75) were very similar to those of native CPO. rCPO catalyzes the oxidation of various substrates in comparable yields and selectivities to native CPO. Indole was oxidized to 2-oxindole with 99% selectivity and thioanisole to the corresponding R-sulfoxide (enantiomeric excess >98%). Incorporation of (18)O from labeled H(2)18O(2) into the oxidized products was 100% in both cases.

Improving the catalytic performance of peroxidases in organic synthesis.

Peroxidases are ubiquitous enzymes that catalyze a variety of enantioselective oxygen-transfer reactions with hydrogen peroxide (H2O2). Although they have enormous potential, their industrial application is hampered by their high price and low operational stability. Recent developments, such as the controlled addition and in situ formation of the oxidant, protein engineering and the rational design of semi-synthetic peroxidases, aim to improve the operational stability of peroxidases.

Highly efficient immobilization of glycosylated enzymes into polyurethane foams.

Glycosylated enzymes, including aminoacylase from Aspergillus melleus, chloroperoxidase from Caldariomyces fumago, and phytase from Aspergillus ficuum, were covalently immobilized into polyurethane foams with very high enzyme loadings of up to 0.2 g protein per gram dry foam. The immobilization efficiency (retained activity) ranged from 100% at a low loading to 60% at high loadings. In contrast to many other immobilization methods no leaching of the enzyme from the support took place under the reaction conditions. In short, a universal method for the immobilization of enzymes from fungal sources was developed, affording a highly active, stable, and reusable biocatalyst.

Biocatalytic and biomimetic oxidations with vanadium.

Approaches to the rational design of vanadium-based semi-synthetic enzymes and biomimetic models as catalysts for enantioselective oxidations are reviewed. Incorporation of vanadate ion into the active site of phytase (E.C. 3.1.3.8), which in vivo mediates the hydrolysis of phosphate esters, afforded a semi-synthetic peroxidase. It catalyzed the enantioselective oxidation of prochiral sulfides with H2O2 affording the S-sulfoxide, e.g. in 66% ee at quantitative conversion of thioanisole. Under the reaction conditions the semi-synthetic vanadium peroxidase was stable for more than 3 days with only a slight decrease in turnover frequency. Amongst the transition-metal oxoanions that are known to be potent inhibitors of phosphatases, only vanadate resulted in a semi-synthetic peroxidase when incorporated into phytase. In a biomimetic approach, vanadium complexes of chiral Schiff base complexes were encapsulated in the super cages of a hydrophobic zeolite Y. Unfortunately, these ship-in-a-bottle complexes afforded only racemic sulfoxide in the catalytic oxidation of thioanisole with H2O2.

Improved operational stability of peroxidases by coimmobilization with glucose oxidase.

The operational stability of peroxidases was considerably enhanced by generating hydrogen peroxide in situ from glucose and oxygen. For example, the total turnover number of microperoxidase-11 in the oxidation of thioanisole was increased sevenfold compared with that obtained with continuous addition of H(2)O(2). Coimmobilization of peroxidases with glucose oxidase into polyurethane foams afforded heterogeneous biocatalysts in which the hydrogen peroxide is formed inside the polymeric matrix from glucose and oxygen. The total turnover number of chloroperoxidase in the oxidation of thioanisole and cis-2-heptene was increased to new maxima of 250. 10(3) and 10. 10(3), respectively, upon coimmobilization with glucose oxidase. Soybean peroxidase, which normally shows only classical peroxidase activity, was transformed into an oxygen-transfer catalyst when coimmobilized with glucose oxidase. The combination catalyst mediated the enantioselective oxidation of thioanisole [50% ee (S)] with 210 catalyst turnovers.

The rational design of semisynthetic peroxidases.

A semisynthetic peroxidase was designed by exploiting the structural similarity of the active sites of vanadium dependent haloperoxidases and acid phosphatases. Incorporation of vanadate ion into the active site of phytase (E.C. 3.1.3.8), which mediates in vivo the hydrolysis of phosphate esters, leads to the formation of a semisynthetic peroxidase, which catalyzes the enantioselective oxidation of prochiral sulfides with H(2)O(2) affording the S-sulfoxide, e.g. in 66% ee at 100% conversion for thioanisole. Under reaction conditions the semi-synthetic vanadium peroxidase is stable for over 3 days with only a slight decrease in turnover frequency. Polar water-miscible cosolvents, such as methanol, dioxane, and dimethoxyethane, can be used in concentrations of 30% (v/v) at a small penalty in activity and enantioselectivity. Among the transition metal oxoanions that are known to be potent inhibitors, only vanadate resulted in a semisynthetic peroxidase when incorporated into phytase. A number of other acid phosphatases and hydrolases were tested for peroxidase activity, when incorporated with vanadate ion. Phytases from Aspergillus ficuum, A. fumigatus, and A. nidulans, sulfatase from Helix pomatia, and phospholipase D from cabbage catalyzed enantioselective oxygen transfer reactions when incorporated with vanadium. However, phytase from A. ficuum was unique in also catalyzing the enantioselective sulfoxidation, albeit at a lower rate, in the absence of vanadate ion.

Nd:YAG laser posterior hyaloidotomy for the treatment of a premacular vitreous floater.

A 63-year old man, with a large central vitreous floater, underwent a Nd:YAG laser posterior hyaloidotomy. Although the vitreous floater disappeared from the central optical axis, visual acuity did not improve. Microperimetry performed with the SLO revealed an absolute scotoma, which corresponded well in shape and dimension with the original vitreous floater. This finding suggests that a fragment of the neurosensory retina became detached together with the internal limiting membrane in the process of the vitreous collapse.

Characterization of functional changes in macular holes and cysts.

Precise characterization of functional loss in small retinal lesions is difficult with conventional techniques. Using the scanning laser ophthalmoscope, the authors evaluated functional changes and fixation behavior in 26 eyes with macular holes and 15 eyes with macular cysts. A dense scotoma was present over all macular holes; 24 had no detectable functional alteration at the margins of the hole, and fixation was above the horizontal meridian in all eyes. Nine eyes with cysts had no detectable functional loss over the cyst. Only two eyes had small areas of dense scotoma within the cyst area, and four had areas of relative scotoma. Fixation was central in all eyes. Characterization of functional changes is helpful in differentiating holes from cysts. Photocoagulation at the margin of the holes may result in further functional damage.

Topographical analysis of peripheral vs central retina with pattern reversal visual evoked response and the scanning laser ophthalmoscope.

The contribution of the central and the surrounding peripheral retina to the pattern reversal visual evoked response (PVER) was analyzed simultaneously using double frequency stimulation and the Fast Fourier transform (FFT) method. The Scanning Laser Ophthalmoscope (SLO) was used to project the pattern stimulus, a square subtending 10 degrees on a side, on a specific location in the fundus and to monitor accurately the stimulus' position during PVER testing. When the stimulus area on the central retina was less than or equal to 50 min of arc on a side, or 0.69% of the total area was stimulated (a square of 10 degrees on a side), the contribution from the central retina was negligible, and the PVER was dominated by surrounding peripheral retinal activity. When the stimulus area of the central retina was greater than or equal to 100 min of arc on a side, or 2.78% of the whole area was stimulated, activity from the central retina became more evident. When the stimulus area projected on the central retina became 150 min on a side (6.25% of the area or greater), activity from the central retina showed an increase and exceeded that of the peripheral retina. When the central area became larger, its activity dominated the PVER, and the contribution from the peripheral retina became negligible at this stage. The PVER was highly dependent upon the activity of the central retina, and activity levels from the central and surrounding peripheral retina were correlated.

Inactive subretinal neovascularization in age-related macular degeneration.

Inactive subretinal new vessels (SRNVs), showing minimal leakage by fluorescein angiography and little progression, were observed in 15 eyes of 12 patients with age-related macular degeneration. In 8 (57%) of 14 eyes followed for longer than 6 months, the new vessels involuted and produced circumscribed areas of retinal pigment epithelium and choriocapillaris atrophy; 6 (43%) showed a further decrease in the fluorescein leakage during the follow-up period (average, 30.1 months). None of the new vessels organized into an exudative fibrovascular scar. Visual acuity was improved or unchanged during the entire follow-up period in 12 (86%) eyes and deteriorated in 2 (14%). Nine patients (75%) were 80 years of age or older; three (25%) were between 65 and 70 years of age. These observations suggested that SRNV showing minimal fluorescein leakage, particularly when it occurs in patients older than 80 years, do not require photocoagulation because they are usually self-limited.

[Static microperimetry with the laser scanning ophthalmoscope]. / La micropérimétrie statique avec l'ophtalmoscope à balayage laser.

We have used the scanning laser opthalmoscope (SLO) with a personal computer to develop static microperimetry techniques. They allow to see in real-time on a television monitor the precise retinal localization of the stimulus and fixation. The testing is performed under strict conditions. The size of stimuli can vary between 6 and 30 minarc on a side. 255 different intensity levels are possible with the instrument. We have selected 12 of them, representing a logarithmic scale. Stimulus duration can vary between 50 and 500 ms. Examples of macular pathology including subretinal neovascularization, drusen and macular edema are given.