Apremilast Is Effective in Lichen Planus Mucosae-Associated Stenotic Esophagitis.

A 74-year-old woman with extensive lichen planus mucosae (LPM) developed stenotic esophagitis that was refractory to intravenous glucocorticosteroids. Esophageal dilatations to 14 mm width were repeatedly performed without any lasting effect. After introducing oral apremilast, she experienced complete clinical remission within the first 4 weeks of treatment. Control esophagoscopy confirmed a marked recovery of the esophageal mucosa with no recurrence of the former stenosis. Our observation is in line with the case series of Paul et al. [J Am Acad Dermatol 2013;68: 255-261] who first reported on the benefit of apremilast in patients with extensive LPM. Ideally, the effectiveness of apremilast in LPM should be studied in a randomized controlled trial.

Structural brain correlates of delay of gratification in the elderly.

Delay of gratification (DoG) refers to the ability to postpone immediate rewards in favor of later and better rewards. A successful DoG in children/adolescents is subject to the maturation of the lateral and medial prefrontal cortex, which is more prone to normal age-related atrophy compared with other brain regions. Therefore, we investigated morphological brain correlates of DoG using structural MRI surface-based morphometry as well as determined whether dorsolateral prefrontal cortex (DLPFC) atrophy is related to DoG in the elderly. We used the behavioral Delay of Gratification Test for Adults to measure DoG in 40 healthy older adults aged between 63 and 93 years. When simultaneously controlling for age and intracranial volume, high DoG significantly positively correlated with cortical surface area of the left DLPFC. At a more liberal statistical threshold, we found positive correlations between DoG and cortical thickness of the left and right DLPFC, left and right ventrolateral prefrontal cortex, and left midanterior cingulate cortex. Additionally, cortical surface area in the left DLPFC correlated positively with DoG as well as with the volume of the left caudate nucleus. The results suggest that the DLPFC, medial prefrontal cortex, and the caudate nucleus play a crucial role in DoG in the elderly supporting studies in related constructs such as delay discounting and impulsivity. Further, the study shows that age-related prefrontal atrophy is associated with DoG performance. The findings are in line with concepts of "willpower" that postulate a central role of frontostriatal connectivity in self-regulation and self-control.

Feline pancreatic islet-like clusters and insulin producing cells express functional Toll-like receptors (TLRs).

Toll-like receptors (TLRs) are cellular receptors that recognize molecules derived from pathogens, endogenous molecules generated after cellular stress, and free fatty acids. TLR activation leads to a proinflammatory reaction that is fundamental in the initiation of an innate immune response and subsequent adaptive responses but also can damage tissues. TLRs are not only expressed within the immune system, but also in most other organ systems including the pancreas. TLR4 is expressed in pancreatic ß-cells of rodents and humans and its stimulation affects insulin secretion in response to glucose. A low-grade inflammation is often associated with disturbed performance of ß-cells and insulin resistance, the cardinal metabolic event of type-2 diabetes. Feline diabetes mellitus shares many similarities with type-2 diabetes in humans. Our objective was to elucidate the role of TLRs in feline pancreatic islets and islet-like clusters (ILC) that consist of islets with their adjacent tissue. We tested whether TLRs are triggered by their agonists and lead to the expression of inflammatory cytokines. We confirmed the expression of all known feline TLRs in pancreas and ILC. Furthermore, stimulation with TLR agonists increased IL-6 mRNA and protein content and the expression of other proinflammatory cytokines indicating a clear proinflammatory response. The reactivity to TLR ligands was stronger in ß-cell enriched populations obtained after sorting by FACS indicating that inflammatory stimuli can also be generated within ß-cells. We conclude that the microenvironment of feline ß-cells harbor the potential for inflammatory reactions, that can be initiated by molecules released from bacteria or viruses or other molecules recognized by TLRs. Therefore infections associated with bacteriemia and viremia can induce inflammation in islets and damage the endocrine pancreatic tissue.

Quantitative real-time PCR detection of insulin signalling-related genes in pancreatic islets isolated from healthy cats.

The cat has recently been proposed as a valuable model for type 2 diabetes mellitus (T2DM), because feline diabetes shares several similarities with the disease in humans. Impaired beta-cell function, decreased beta-cell mass, insulin resistance that is often related to obesity, and pancreatic amyloid deposition, are among these common features. In this study, and to further develop the cat as a model of T2DM, feline pancreatic islets were isolated and real-time PCR quantification of mRNA transcripts of genes central to beta-cell function and survival established. In particular, mRNA quantification systems were determined for insulin, the insulin enhancer pancreatic duodenal homeobox-1 (PDX-1), the insulin suppressor CCAAT/enhancer binding protein-beta (C/EBPbeta), glucose transporter isoform 2 (GLUT2), Fas receptor, the caspase-8 inhibitor FLIP (FLICE [caspase-8]-inhibitory protein) and two chemokines, interleukin (IL)-8 and monocyte chemoattractant protein-1 (MCP-1). Pancreatic islets were isolated by collagenase digestion from healthy cat donors. Partial feline mRNA sequences were determined for PDX-1, C/EBPbeta, GLUT2 and FLIP using primers identified from conserved regions of human, dog and rat mRNA. These novel and the previously available sequences (insulin, Fas receptor, IL-8 and MCP-1) were used to design feline-specific primers suitable for real-time PCR in isolated pancreatic islets. The adopted protocol of collagenase digestion yielded pancreatic islets that were frequently surrounded by acinar cells. Quantification of mRNA transcripts was simple and reproducible in healthy cats. Characterisation of genes related to insulin signalling in cats will prove useful to better understand the pathogenesis of feline diabetes and possibly of human T2DM.

The dipeptidyl peptidase IV inhibitor NVP-DPP728 reduces plasma glucagon concentration in cats.

Glucagon-like peptide-1 (GLP-1) analogues and inhibitors of its degrading enzyme, dipeptidyl peptidase IV (DPPIV), are interesting therapy options in human diabetics because they increase insulin secretion and reduce postprandial glucagon secretion. Given the similar pathophysiology of human type 2 and feline diabetes mellitus, this study investigated whether the DPPIV inhibitor NVP-DPP728 reduces plasma glucagon levels in cats. Intravenous glucose tolerance tests (ivGTT; 0.5 g/kg glucose after 12 h fasting) and a meal response test (test meal of 50% of average daily food intake, offered after 24 h fasting) were performed in healthy experimental cats. NVP-DPP728 (0.5-2.5 mg/kg i.v. or s.c.) significantly reduced glucagon output in all tests and increased insulin output in the ivGTT. Follow-up studies will investigate the potential usefulness as therapy in diabetic cats.

Assessment of six different collagenase-based methods to isolate feline pancreatic islets.

Isolation of pancreatic islets is necessary to study the molecular mechanisms underlying beta-cell demise in diabetic cats. Six collagenase-based methods of isolation were compared in 10 cat pancreata, including single and double course of collagenase, followed or not by Ficoll centrifugation or accutase, and collagenase plus accutase. Morphometric analysis was performed to measure the relative area of islet and exocrine tissue. Islet specific mRNA transcripts were quantified in isolates by real-time PCR. The single and double course of collagenase digestion was successful in each cat and provided similar islet-to-exocrine tissue ratio. Quantities of insulin mRNA did not differ between the two methods. However, on histological examination either method yielded only approximately 2% of pure islets. The other methods provided disrupted islets or insufficient samples in 1-7 cats. Although pancreas digestion with single and double course of collagenase was superior, further studies are needed to improve islet isolation in cats.

Partial sequencing and expression of genes involved in glucose metabolism in adipose tissues and skeletal muscle of healthy cats.

Impaired insulin sensitivity is increasingly recognised in cats, but sequences of genes involved in insulin-signalling are largely undetermined in this species. In this study, extended feline mRNA sequences were determined for the adiponectin, glucose transporter-1 (GLUT1), GLUT4, peroxisome proliferative activated receptor-gamma1 (PPARgamma1), PPARgamma2, plasminogen activator inhibitor-1 (PAI-1), monocyte chemoattractant protein-1 (MCP-1) and insulin receptor genes. Conserved dog-specific primers identified from human-dog mRNA alignments were used to amplify feline cDNA in the polymerase chain reaction (PCR). The feline sequences determined by this method were used to design feline-specific primers suitable for real-time PCR for quantification of gene expression in insulin sensitive tissues of healthy cats. Partial sequences of feline mRNAs had 86-95% identity with dog and human genes. Expression of adiponectin, GLUT1, GLUT4, PPARgamma1, PPARgamma2, PAI-1 and insulin receptor mRNA was detected and quantified in subcutaneous and visceral fat and skeletal muscle, whereas MCP-1 mRNA was detected in adipose tissue but not in skeletal muscle. Further characterisation of genes related to glucose metabolism in cats will provide additional insights into insulin-signalling mechanisms in this species.

Analytical validation of commercial immunoassays for the measurement of cardiovascular peptides in the dog.

Immunoassays for the measurement of concentrations of the cardiovascular peptides pro-atrial natriuretic peptide (proANP), brain natriuretic peptide (BNPPen and BNPPhoe), endothelin-1 (ET-1Bio, ET-1IBL and ET-1Phoe) and big endothelin-1 (Big-ETBio and Big-ETIBL) were validated in canine serum by determination of intra-assay variability and dilutional parallelism. Commercial kits that showed good results were further validated by determination of intra- and inter-assay variability, dilutional parallelism and spiking recovery. Assays for proANP, BNPPhoe, ET-1IBL and Big-ETIBL showed acceptable results in the preliminary validation and were fully validated. The intra- and inter-assay variability was acceptable for all four assays, linearity was demonstrated and recovery rates were acceptable. The performances of the different immunoassays varied considerably, underscoring the importance of validation. Of the assays studied, proANP, BNP(Phoe), ET-1IBL and Big-ETIBL produced precise, reproducible and accurate results and can be recommended for clinical application.

Monitoring survival and function of transfused platelets in Bernard-Soulier syndrome by flow cytometry and a cone and plate(let) analyzer (Impact-R).

BACKGROUND: Bernard-Soulier syndrome (BSS) patients may repeatedly require transfusion of platelets (PLTs). The hemostatic competence of transfused PLTs requires monitoring. STUDY DESIGN AND METHODS: Flow cytometry and a cone and plate(let) analyzer (Impact-R, DiaMed) were used to monitor survival and function of transfused PLTs in a 7-year-old girl with BSS undergoing surgery. Flow cytometry was applied to differentiate autologous PLTs from transfused PLTs by staining for CD42b. The Impact, which measures PLT adhesion and aggregation in response to high shear stress, was used to evaluate PLT function. RESULTS: Transfused PLTs were detectable by flow cytometry for 1 week after transfusion. While the patient's PLTs did not respond to high shear stress before transfusion, a normal response was documented by the Impact on the day after transfusion and 1 week thereafter. CONCLUSION: Transfused PLTs were detectable by flow cytometry, and their functional activity was demonstrated by the Impact.

Effects of ingestion of tomatoes, tomato juice and tomato purée on contents of lycopene isomers, tocopherols and ascorbic acid in human plasma as well as on lycopene isomer pattern.

Tomatoes are an important part of the diet. Lycopene, the predominant carotenoid in tomatoes, is hypothesised to mainly mediate the health benefits of tomato products. Anticancer activity of tomato products and lycopene has been suggested by numerous studies. The aim of the present study was to investigate the effect of ingestion of three different tomato-based foodstuffs on plasma contents of lycopene, tocopherols and ascorbic acid. Because isomers of lycopene may have different biological activities, a special interest was to look how the lycopene isomer pattern is changed depending on the matrix of tomato products. Following a 2-week depletion phase volunteers ingested 12.5 mg lycopene/d for 4 weeks comprising tomatoes, tomato juice or tomato purée. The basal levels of lycopene in plasma were comparable for all groups and decreased significantly during the 2 weeks of depletion to approximately half of the basal values. Following intervention, plasma lycopene concentration increased significantly. Conversely, supplementation did not significantly affect levels of tocopherols and ascorbic acid in plasma. Regarding isomers of lycopene, the (Z)-lycopene:(all-E)-lycopene plasma isomer ratio was significantly changed during the study for all groups. A remarkable enrichment of the relative contents of (5Z)-lycopene was observed during the depletion period, which supports the hypothesis that lycopene (Z)-isomers are formed within the human body after ingestion of (all-E)-lycopene. After dietary intervention with lycopene-rich products the isomer ratios returned to those observed at the start of the study. Further investigations will clarify the process of isomerisation in more detail.

Miniaturized test system for soil respiration induced by volatile pollutants.

A miniaturized method based on 96-well microtitre plates was developed and used to study respiration in pristine and contaminated soils following addition of volatile substrates. Small soil samples were exposed to fuel components, which were volatilized from spatially separate reservoirs of 2,2,4,4,6,8,8-heptamethylnonane (HMN) as an organic carrier. Respiration was determined as CO(2) production by means of a pH-indicator and bicarbonate-containing agar, or as (14)CO(2) evolution from (14)C-labelled substrates. Substrate concentrations inducing maximum microbial activity or inhibition were determined and CO(2) production profiles examined by multivariate analysis. When high concentrations of fuel components were applied, distinction of hydrocarbon exposed soils from unexposed soil was achieved within 6 h of incubation. With low concentrations, adequate distinction was achieved after 24 h, probably as a result of community adaptation. Nutrient limitation was identified with the (14)C method for toluene, and the optimal N and P amendment determined. Further potential applications of this rapid and inexpensive method are outlined.

Microbial community response to petroleum hydrocarbon contamination in the unsaturated zone at the experimental field site Vaerløse, Denmark.

This study investigates the influence of petroleum hydrocarbons on a microbial community in the vadose zone under field conditions. An artificial hydrocarbon mixture consisting of volatile and semi-volatile compounds similar to jet-fuel was emplaced in a previously uncontaminated vadose zone in nutrient-poor glacial melt water sand. The experiment included monitoring of microbial parameters and CO(2) concentrations in soil gas over 3 months in and outside the hydrocarbon vapor plume that formed around the buried petroleum. Microbial and chemical analyses of soil and vadose zone samples were performed on samples from cores drilled to 3.3 m depth on three dates and three lateral distances from the buried petroleum mass. Significantly elevated CO(2) concentrations were observed after contamination. Total cell numbers as determined by fluorescence microscopy were strongly correlated with soil organic carbon and nitrogen content but varied little with contamination. Redundancy analysis (RDA) allowed direct analysis of effects of selected environmental variables or the artificial contamination on microbiological parameters. Variation in biomass and CO(2) production was explained by soil parameters, to 46%, and by the duration of contamination, to 39.8%. The microbial community structure was assessed by community level physiological profiles (CLPP) analysis using Biolog(TM) Eco-Plates. In the CLPP data only 35.9% of the variation could be linked to soil parameters and contamination, however, the samples with greatest exposure to hydrocarbons grouped together on RDA plots. It is concluded that, at this nutrient-poor site, the microbial community was dominated by natural heterogeneity and that the influence of petroleum hydrocarbon vapors was weak.

Impacts of heavy metal contamination and phytoremediation on a microbial community during a twelve-month microcosm experiment.

The effects of heavy metals and phytoextraction practices on a soil microbial community were studied during 12 months using a hyperaccumulating plant (Thlaspi caerulescens) grown in an artificially contaminated soil. The 16S ribosomal RNA genes of the Bacteria and the beta-Proteobacteria and the amoA gene (encoding the alpha-subunit of ammonia monooxygenase) were PCR-amplified and analysed by denaturing gradient gel electrophoresis (DGGE). Principal component analysis (PCA) of the DGGE data revealed that: (i) the heavy metals had the most drastic effects on the bacterial groups targeted, (ii) the plant induced changes which could be observed in the amoA and in the Bacteria 16S rRNA gene patterns, (iii) the changes observed during 12 months in the DGGE-patterns of the planted contaminated soil did not indicate recovery of the initial bacterial community present in the non-contaminated soil. The potential function of the microbial community was assessed recording community level physiological profiles (CLPP) and analysing them by PCA. The lower capability of the bacterial community to degrade the substrates provided in the BIOLOG plates, in particular the amino acids, amides and amines, as well as a delay in the average well colour development (AWCD) differentiated the bacterial community of the contaminated samples from that of the non-contaminated ones. However, the plant had a positive effect on substrate utilization as shown by the greater number of substrates used in all planted samples compared to unplanted ones. Finally, the measurement of the potential ammonia oxidation indicated that ammonia oxidising bacteria were completely inhibited in the contaminated soil. The stimulation of ammonia oxidation by the plant observed in the non-contaminated samples was surpassed by the inhibitory effect of the heavy metals in the contaminated soil. This study emphasises the combined use of culture-independent techniques with conventional methods to investigate the ecology of bacteria in their natural habitats.

Biodegradation of petroleum hydrocarbon vapors: laboratory studies on rates and kinetics in unsaturated alluvial sand.

Predictions of natural attenuation of volatile organic compounds (VOCs) in the unsaturated zone rely critically on information about microbial biodegradation kinetics. This study aims at determining kinetic rate laws for the aerobic biodegradation of a mixture of 12 volatile petroleum hydrocarbons and methyl tert-butyl ether (MTBE) in unsaturated alluvial sand. Laboratory column and batch experiments were performed at room temperature under aerobic conditions, and a reactive transport model for VOC vapors in soil gas coupled to Monod-type degradation kinetics was used for data interpretation. In the column experiment, an acclimatization of 23 days took place before steady-state diffusive vapor transport through the horizontal column was achieved. Monod kinetic parameters Ks and vmax could be derived from the concentration profiles of toluene, m-xylene, n-octane, and n-hexane, because substrate saturation was approached with these compounds under the experimental conditions. The removal of cyclic alkanes, isooctane, and 1,2,4-trimethylbenzene followed first-order kinetics over the whole concentration range applied. MTBE, n-pentane, and chlorofluorocarbons (CFCs) were not visibly degraded. Batch experiments suggested first-order disappearance rate laws for all VOCs except n-octane, which decreased following zero-order kinetics in live batch experiments. For many compounds including MTBE, disappearance rates in abiotic batch experiments were as high as in live batches indicating sorption. It was concluded that the column approach is preferable for determining biodegradation rate parameters to be used in risk assessment models.

Vapor phase transport and biodegradation of volatile fuel compounds in the unsaturated zone: a large scale lysimeter experiment.

The vapor phase transport and biodegradation of typical fuel compounds including volatile petroleum hydrocarbons and methyl tert-butyl ether (MTBE) was studied in a large scale field lysimeter representing a 2.3 m thick sandy unsaturated zone over a gravel aquifer. A mixture of 13 fuel compounds with MTBE (5%) was placed at a defined depth in the unsaturated zone to obtain a homogeneous source zone with a residual NAPL saturation. The upward and downward transport of fuel vapors and the biodegradation by indigenous microorganisms were monitored during 70 days. Using tracers in water and NAPL, it was shown that fuel compounds were transported by vapor phase diffusion only. All fuel compounds except MTBE disappeared from the lysimeter below the analytical detection limits within 70 days. MTBE accumulated in groundwater but volatilized from the unsaturated zone. First-order biodegradation rates were estimated in the unsaturated zone to range from <0.05 d(-1) for MTBE up to 8.7 d(-1) for octane. Aerobic biodegradation of degradable fuel compounds to CO2 started without any lag phase and removed about 3 times more fuel mass than volatilization. The study illustrates the recalcitrance of MTBE vapors compared to other fuel vapors, leading to a significant groundwater pollution with MTBE.