Pre-transplant signal induction for vascularisation in human islets

Human islet transplant success is partially impaired by slow revascularisation. Our study investigated the potential for rotational cell culture (RC) of human islets combined with thiazolidinedione (TZD) stimulation of peroxisome proliferator-activated receptor gamma (PPAR?) to upregulate vascular endothelial growth factor (VEGF) expression in the islets. Four groups of human islets were studied: static culture (SC) with and without 25 mmol/L TZD and RC with and without 25 mmol/L TZD. These were assessed for insulin secretion and soluble VEGF-A release. Both proteins were quantified by enzyme-linked immunosorbent assay (ELISA), supported with qualitative immunofluorescence staining. RC + TZD increased insulin secretion by >20% (p <0.05-0.001) in response to 16.7 mmol/L glucose and 16.7 mmol/L glucose + 10 mmol/L theophylline (G + T). This effect was seen at all time intervals compared with SC and without addition of TZD. Soluble VEGF-A release was significantly augmented by RC and TZD exposure with an increased effect of >30% (p <0.001) at 72 h under both SC and RC conditions. RC supplemented with a TZD enhances and prolongs the release of insulin and soluble VEGF-A by isolated human islets. © 2013 The Author(s).

Modulation of interleukin-6 and matrix metalloproteinase 2 expression in human fibroblast-like synoviocytes by functional ionotropic glutamate receptors

Objective. Patients with rheumatoid arthritis (RA) have increased concentrations of the amino acid glutamate in synovial fluid. This study was undertaken to determine whether glutamate receptors are expressed in the synovial joint, and to determine whether activation of glutamate receptors on human synoviocytes contributes to RA disease pathology. Methods. Glutamate receptor expression was examined in tissue samples from rat knee joints and in human fibroblast-like synoviocytes (FLS). FLS from 5 RA patients and 1 normal control were used to determine whether a range of glutamate receptor antagonists influenced expression of the proinflammatory cytokine interleukin-6 (IL-6), enzymes involved in matrix degradation and cytokine processing (matrix metalloproteinase 2 [MMP-2] and MMP-9), and the inhibitors of these enzymes (tissue inhibitor of metalloproteinases 1 [TIMP-1] and TIMP-2). IL-6 concentrations were determined by enzyme-linked immunosorbent assay, MMP activity was measured by gelatin zymography, and TIMP activity was determined by reverse zymography. Fluorescence imaging of intracellular calcium concentrations in live RA FLS stimulated with specific antagonists was used to reveal functional activation of glutamate receptors that modulated IL-6 or MMP-2. Results. Ionotropic and metabotropic glutamate receptor subunit mRNA were expressed in the patella, fat pad, and meniscus of the rat knee and in human articular cartilage. Inhibition of N-methyl-D-aspartate (NMDA) receptors in RA FLS increased proMMP-2 release, whereas non-NMDA ionotropic glutamate receptor antagonists reduced IL-6 production by these cells. Stimulation with glutamate, NMDA, or kainate (KA) increased intracellular calcium concentrations in RA FLS, demonstrating functional activation of specific ionotropic glutamate receptors. Conclusion. Our findings indicate that activation of NMDA and KA glutamate receptors on human synoviocytes may contribute to joint destruction by increasing IL-6 expression. © 2007, American College of Rheumatology.

Antiangiogenic effect of soluble vascular endothelial growth factor receptor-1 in placental angiogenesis

Differential splicing of the flt-1 mRNA generates soluble variant of vascular endothelial growth factor (VEGF) receptor-1 (sVEGFR-1, also known as sFlt-1). The action of VEGF is antagonized by sVEGFR-1. Soluble VEGFR-1 binds to VEGF with a high affinity and therefore works to modulate VEGF and VEGF signaling pathway. In this study, the authors tested the hypothesis that VEGF-mediated endothelial cell angiogenesis is tightly modulated by the release of sVEGFR-1 and placental expression of sVEGFR-1 is upregulated by hypoxia. Immunolocalization studies showed progressively intense staining for sVEGFR-1 and VEGF in the trophoblast of placental villous explants throughout gestation. Endothelial cell migration studies using a modified Boyden's chamber showed a significant increase in cell migration in response to VEGF that was significantly attenuated in the presence of exogenous sVEGFR-1. Furthermore, stimulation of endothelial cells with VEGF led to a dose-dependent increase in the release of sVEGFR-1 as determined by enzyme-linked immunosorbent assay (ELISA). Exposure of normal placental villous explants to hypoxia (1% pO2) increased trophoblast expression of sVEGFR-1 when compared with tissue normoxia (5% pO2). In addition, conditioned media from hypoxia treated placental villous explants induced a significant increase in endothelial cell migration that was significantly reduced in presence of sVEGFR-1. Our study demonstrates that hypoxia positively regulates sVEGFR-1 protein expression in ex vivo trophoblasts, which control VEGF-driven angiogenesis.

Resveratrol inhibits the release of soluble fms-like tyrosine kinase (sFlt-1) from human placenta

Objective - Soluble vascular endothelial growth factor receptor–1 (also know as soluble fms-like tyrosine kinase [sFlt]-1) is a key causative factor of preeclampsia. Resveratrol, a plant phytoalexin, has antiinflammatory and cardioprotective properties. We sought to determine the effect of resveratrol on sFlt-1 release. Study Design - Human umbilical vein endothelial cells, transformed human trophoblast-8 (HTR/SVneo)-8/SVneo trophoblast cells, or placental explants were incubated with cytokines and/or resveratrol. Conditioned media were assayed for sFlt-1 by enzyme-linked immunosorbent assay and cell proteins used for Western blotting. Results - Resveratrol inhibited cytokine-induced release of sFlt-1 from normal placental explants and from preeclamptic placental explants. Preincubation of human umbilical vein endothelial cells or HTR-8/SVneo cells with resveratrol abrogated sFlt-1 release. Resveratrol prevented the up-regulation of early growth response protein-1 (Egr-1), a transcription factor necessary for induction of the vascular endothelial growth factor receptor–1 gene and caused up-regulation of heme oxygenase–1, a cytoprotective enzyme found to be dysfunctional in preeclampsia. Conclusion - In summary, resveratrol can inhibit sFlt-1 release and up-regulate heme oxygenase–1; thus, may offer therapeutic potential in preeclampsia.

A rapid ELISA for the diagnosis of intravascular catheter related sepsis caused by coagulase negative staphylococci

Aim: To develop and evaluate a rapid enzyme linked immunosorbent assay (ELISA) for the diagnosis of intravascular catheter related sepsis caused by coagulase negative staphylococci. Methods: Forty patients with a clinical and microbiological diagnosis of intravascular catheter related sepsis and positive blood cultures, caused by coagulase negative staphylococci, and 40 control patients requiring a central venous catheter as part of their clinical management were recruited into the study. Serum IgG responses to a previously undetected exocellular antigen produced by coagulase negative staphylococci, termed lipid S, were determined in the patient groups by a rapid ELISA. Results: There was a significant difference (p = < 0.0001) in serum IgG to lipid S between patients with catheter related sepsis and controls. The mean antibody titre in patients with sepsis caused by coagulase negative staphylococci was 10 429 (range, no detectable serum IgG antibody to 99 939), whereas serum IgG was not detected in the control group of patients. Conclusions: The rapid ELISA offers a simple, economical, and rapid diagnostic test for suspected intravascular catheter related sepsis caused by coagulase negative staphylococci, which can be difficult to diagnose clinically. This may facilitate treatment with appropriate antimicrobials and may help prevent the unnecessary removal of intravascular catheters.

Ankyrin is the major oxidised protein in erythrocyte membranes from end-stage renal disease patients on chronic haemodialysis and oxidation is decreased by dialysis and vitamin C supplementation

Chronically haemodialysed end-stage renal disease patients are at high risk of morbidity arising from complications of dialysis, the underlying pathology that has led to renal disease and the complex pathology of chronic kidney disease. Anaemia is commonplace and its origins are multifactorial, involving reduced renal erythropoietin production, accumulation of uremic toxins and an increase in erythrocyte fragility. Oxidative damage is a common risk factor in renal disease and its co-morbidities and is known to cause erythrocyte fragility. Therefore, we have investigated the hypothesis that specific erythrocyte membrane proteins are more oxidised in end-stage renal disease patients and that vitamin C supplementation can ameliorate membrane protein oxidation. Eleven patients and 15 control subjects were recruited to the study. Patients were supplemented with 2 × 500 mg vitamin C per day for 4 weeks. Erythrocyte membrane proteins were prepared pre- and post-vitamin C supplementation for determination of protein oxidation. Total protein carbonyls were reduced by vitamin C supplementation but not by dialysis when investigated by enzyme linked immunosorbent assay. Using a western blot to detect oxidised proteins, one protein band, later identified as containing ankyrin, was found to be oxidised in patients but not controls and was reduced significantly by 60% in all patients after dialysis and by 20% after vitamin C treatment pre-dialysis. Ankyrin oxidation analysis may be useful in a stratified medicines approach as a possible marker to identify requirements for intervention in dialysis patients.

Monocyte subpopulation counts and TNF alpha associations with clinical outcome post ST elevation myocardial infarction

Background Monocytes are implicated in the initiation and progression of the atherosclerotic plaque contributing to its instability and rupture. Although peripheral monocytosis has been related to poor clinical outcome post ST elevation myocardial infarction (STEMI), only scarce information is available of mechanisms of this association. Tumour necrosis factor alpha (TNFα) is a key cytokine in the acute phase inflammatory response, and it is predominantly produced by inflammatory macrophages. Little is known about TNFα association with circulating monocyte subpopulations post STEMI. Method A total of 142 STEMI patients (mean age 62±13 years; 72% male) treated with percutaneous revascularization were recruited with blood samples obtained within first 24 hours from the onset and on day 10-14. Peripheral blood monocyte subpopulations were enumerated and characterized using flow cytometry after staining for CD14, CD16 and CCR2 and were defined as: CD14++CD16-CCR2+ (Mon1), CD14++CD16+CCR+ (Mon2) and CD14+CD16++CCR2- (Mon3) cells. Plasma levels of TNFα were measured by enzyme-linked immunosorbent assay (ELISA, Peprotec system, UK). Major adverse cardiac events (MACE), defined as recurrent STEMI, new diagnosis of heart failure and death were recorded at follow up, mean of 164±134 days. Results TNFα levels were significantly higher 24 hours post STEMI, compared to day 14 (paired t-test, p <0.001) with day 1 levels weakly correlated with total monocyte count as well as Mon1 (Spearman’s correlation, r=0.19, p=0.02 and r=0.22, p=0.01, respectively). There was no correlation between TNFα and Mon2 or Mon3 subpopulations. TNFα levels were significantly higher in patients with a recorded MACE (n=28, Mann-Whitney test, p<0.001) (figure 1).⇓

Synthesis, Modification, and Application of Siloxane Materials for Environmental Sensing

As human populations and resource consumption increase, it is increasingly important to monitor the quality of our environment. While laboratory instruments offer useful information, portable, easy to use sensors would allow environmental analysis to occur on-site, at lower cost, and with minimal operator training. We explore the synthesis, modification, and applications of modified polysiloxane in environmental sensing. Multiple methods of producing modified siloxanes were investigated. Oligomers were formed by using functionalized monomers, producing siloxane materials containing silicon hydride, methyl, and phenyl side chains. Silicon hydride-functionalized oligomers were further modified by hydrosilylation to incorporate methyl ester and naphthyl side chains. Modifications to the siloxane materials were also carried out using post-curing treatments. Methyl ester-functionalized siloxane was incorporated into the surface of a cured poly(dimethylsiloxane) film by siloxane equilibration. The materials containing methyl esters were hydrolyzed to reveal carboxylic acids, which could later be used for covalent protein immobilization. Finally, the siloxane surfaces were modified to incorporate antibodies by covalent, affinity, and adsorption-based attachment. These modifications were characterized by a variety of methods, including contact angle, attenuated total reflectance Fourier transform infrared spectroscopy, dye labels, and 1H nuclear magnetic resonance spectroscopy. The modified siloxane materials were employed in a variety of sensing schemes. Volatile organic compounds were detected using methyl, phenyl, and naphthyl-functionalized materials on a Fabry-Perot interferometer and a refractometer. The Fabry-Perot interferometer was found to detect the analytes upon siloxane extraction by deformation of the Bragg reflectors. The refractometer was used to determine that naphthyl-functionalized siloxanes had elevated refractive indices, rendering these materials more sensitive to some analytes. Antibody-modified siloxanes were used to detect biological analytes through a solid phase microextraction-mediated enzyme linked immunosorbent assay (SPME ELISA). The SPME ELISA was found to have higher analyte sensitivity compared to a conventional ELISA system. The detection scheme was used to detect Escherichia coli at 8500 CFU/mL. These results demonstrate the variety of methods that can be used to modify siloxanes and the wide range of applications of modified siloxanes has been demonstrated through chemical and biological sensing schemes.

Distribution, occurrence and biotoxin composition of the main shellfish toxin producing microalgae within European waters: A comparison of methods of analysis.

Harmful algal blooms (HABs) are a natural global phenomena emerging in severity and extent. Incidents have many economic, ecological and human health impacts. Monitoring and providing early warning of toxic HABs are critical for protecting public health. Current monitoring programmes include measuring the number of toxic phytoplankton cells in the water and biotoxin levels in shellfish tissue. As these efforts are demanding and labour intensive, methods which improve the efficiency are essential. This study compares the utilisation of a multitoxin surface plasmon resonance (multitoxin SPR) biosensor with enzyme-linked immunosorbent assay (ELISA) and analytical methods such as high performance liquid chromatography with fluorescence detection (HPLC-FLD) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) for toxic HAB monitoring efforts in Europe. Seawater samples (n = 256) from European waters, collected 2009–2011, were analysed for biotoxins: saxitoxin and analogues, okadaic acid and dinophysistoxins 1/2 (DTX1/DTX2) and domoic acid responsible for paralytic shellfish poisoning (PSP), diarrheic shellfish poisoning (DSP) and amnesic shellfish poisoning (ASP), respectively. Biotoxins were detected mainly in samples from Spain and Ireland. France and Norway appeared to have the lowest number of toxic samples. Both the multitoxin SPR biosensor and the RNA microarray were more sensitive at detecting toxic HABs than standard light microscopy phytoplankton monitoring. Correlations between each of the detection methods were performed with the overall agreement, based on statistical 2 × 2 comparison tables, between each testing platform ranging between 32% and 74% for all three toxin families illustrating that one individual testing method may not be an ideal solution. An efficient early warning monitoring system for the detection of toxic HABs could therefore be achieved by combining both the multitoxin SPR biosensor and RNA microarray.

Distribution, occurrence and biotoxin composition of the main shellfish toxin producing microalgae within European waters: A comparison of methods of analysis.

Harmful algal blooms (HABs) are a natural global phenomena emerging in severity and extent. Incidents have many economic, ecological and human health impacts. Monitoring and providing early warning of toxic HABs are critical for protecting public health. Current monitoring programmes include measuring the number of toxic phytoplankton cells in the water and biotoxin levels in shellfish tissue. As these efforts are demanding and labour intensive, methods which improve the efficiency are essential. This study compares the utilisation of a multitoxin surface plasmon resonance (multitoxin SPR) biosensor with enzyme-linked immunosorbent assay (ELISA) and analytical methods such as high performance liquid chromatography with fluorescence detection (HPLC-FLD) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) for toxic HAB monitoring efforts in Europe. Seawater samples (n = 256) from European waters, collected 2009–2011, were analysed for biotoxins: saxitoxin and analogues, okadaic acid and dinophysistoxins 1/2 (DTX1/DTX2) and domoic acid responsible for paralytic shellfish poisoning (PSP), diarrheic shellfish poisoning (DSP) and amnesic shellfish poisoning (ASP), respectively. Biotoxins were detected mainly in samples from Spain and Ireland. France and Norway appeared to have the lowest number of toxic samples. Both the multitoxin SPR biosensor and the RNA microarray were more sensitive at detecting toxic HABs than standard light microscopy phytoplankton monitoring. Correlations between each of the detection methods were performed with the overall agreement, based on statistical 2 × 2 comparison tables, between each testing platform ranging between 32% and 74% for all three toxin families illustrating that one individual testing method may not be an ideal solution. An efficient early warning monitoring system for the detection of toxic HABs could therefore be achieved by combining both the multitoxin SPR biosensor and RNA microarray.

Proteomic analysis of coronary sinus serum reveals leucine-rich α2-glycoprotein as a novel biomarker of ventricular dysfunction and heart failure

BACKGROUND: Heart failure (HF) prevention strategies require biomarkers that identify disease manifestation. Increases in B-type natriuretic peptide (BNP) correlate with increased risk of cardiovascular events and HF development. We hypothesize that coronary sinus serum from a high BNP hypertensive population reflects an active pathological process and can be used for biomarker exploration. Our aim was to discover differentially expressed disease-associated proteins that identify patients with ventricular dysfunction and HF.

METHODS AND RESULTS: Coronary sinus serum from 11 asymptomatic, hypertensive patients underwent quantitative differential protein expression analysis by 2-dimensional difference gel electrophoresis. Proteins were identified using mass spectrometry and then studied by enzyme-linked immunosorbent assay in sera from 40 asymptomatic, hypertensive patients and 105 patients across the spectrum of ventricular dysfunction (32 asymptomatic left ventricular diastolic dysfunction, 26 diastolic HF, and 47 systolic HF patients). Leucine-rich α2-glycoprotein (LRG) was consistently overexpressed in high BNP serum. LRG levels correlate significantly with BNP in hypertensive, asymptomatic left ventricular diastolic dysfunction, diastolic HF, and systolic HF patient groups (P≤0.05). LRG levels were able to identify HF independent of BNP. LRG correlates with coronary sinus serum levels of tumor necrosis factor-α (P=0.009) and interleukin-6 (P=0.021). LRG is expressed in myocardial tissue and correlates with transforming growth factor-βR1 (P<0.001) and α-smooth muscle actin (P=0.025) expression.

CONCLUSIONS: LRG was identified as a serum biomarker that accurately identifies patients with HF. Multivariable modeling confirmed that LRG is a stronger identifier of HF than BNP and this is independent of age, sex, creatinine, ischemia, β-blocker therapy, and BNP.

The Effect of Dietary Tartrazine on Brain Dopamine and the Behavioral Symptoms of Attention Deficit Hyperactivity Disorder

Attention Deficit Hyperactivity Disorder is a neurodevelopmental disorder correlated with a decrease in brain dopamine and an increase in behavioral symptoms of hyperactivity and impulsivity. This experiment explored how tartrazine (Yellow #5) impacts these symptoms. After tartrazine administration to Spontaneously Hypertensive Rats (SHR), dopamine concentrations in regions of brain tissue were measured using Enzyme-Linked Immunosorbent Assay analysis. Behavioral testing with a T-maze and open field test measured impulsivity and hyperactivity, respectively. Results indicate that dietary tartrazine increases hyperactive behaviors in the SHR. However, results do not indicate a relationship between dietary tartrazine and brain dopamine. No conclusions regarding the relationship between dietary tartrazine and impulsivity were drawn.

Occupational exposure to Aflatoxin B1 in a Portuguese poultry slaughterhouse

Aflatoxin B1 (AFB1) is a secondary metabolite produced by the fungi Aspergillus flavus and is the most potent hepatocarcinogen known in mammals and has been classified by the International Agency of Research on Cancer as Group 1 carcinogen. Although dietary exposure to AFB1 has been extensively documented, there are still few studies dedicated to the problem of occupational exposure. Considering recent findings regarding AFB1 occupational exposure in poultry production, it was considered relevant to clarify if there is also exposure in poultry slaughterhouses. Occupational exposure assessment to AFB1 was done with a biomarker of internal dose that measures AFB1 in the serum by enzyme-linked immunosorbent assay. Thirty workers from a slaughterhouse were enrolled in this study. A control group (n = 30) was also considered in order to know AFB1 background levels for Portuguese population. Fourteen workers (47.0%) showed detectable levels of AFB1 with values from 1.06 to 4.03ng ml(-1), with a mean value of 1.73ng ml(-1). No AFB1 was detected in serum of individuals used as controls. Despite uncertainties regarding the exposure route that is contributing more to exposure (inhalation or dermal) is possible to state that exposure to AFB1 is occurring in the slaughterhouse studied. It seems that reducing AFB1 contamination in poultry production can have a positive result in this occupational setting.

Survival, differentiation, and neuroprotective mechanisms of human stem cells complexed with neurotrophin-3-releasing pharmacologically active microcarriers in an ex vivo model of Parkinson's disease

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Association of interleukin 17 / angiotensin II with refractory hypertension risk in hemodialysis patients.

Objective: The study was performed to investigate the association of interleukin 17 (IL 17) or angiotensin II (Ang II) with refractory hypertension risk in hemodialysis patients. Methods: Ninety hemodialysis patients were enrolled into this study, and those with hypertension were divided into two groups. The Easy-to-Control Hypertension group (ECHG) had fifty patients, while the refractory hypertension group (RHG) had forty patients. Twenty healthy individuals were recruited as the control group. IL17 and Ang II were determined using a human IL 17 / Ang II enzyme-linked immunosorbent assay kit. Serum IL 17 and Ang II concentrations in RHG patients were higher than those in ECHG patients. Results: Serum IL 17 and Ang II concentrations in both patient groups were higher than those in the control group. Linear regression analysis showed a positive correlation between IL 17 and Ang II. In multivariate regression analysis, we found that IL17 and Ang II were associated with refractory hypertension risk in hemodialysis patients. Conclusion: IL17 and Ang II were associated with refractory hypertension risk in hemodialysis patients. There was also a positive correlation between IL 17and Ang II.