A study of potential serum markers for a diagnosis of gram-positive and gram-negative bacterial sepsis

Sepsis continues to be a major cause of morbidity and mortality as it can readily lead tosevere sepsis, septic shock, multiple organ failure and death. The onset can be rapid and difficult to define clinically. Despite the numerous candidate markers proposed in the literature, to date a serum marker for sepsis has not been found. The aim of this study was to assay the serum of clinically diagnosed patients with eithera Gram-negative or Gram- positive bacterial sepsis for elevated levels of nine potentialmarkers of sepsis, using commercially produced enzyme linked immunosorbent assays(ELISA). The purpose was to find a test marker for sepsis that would be helpful toclinicians in cases of uncertain sepsis and consequently expose false positive BC'scaused by skin or environmental contaminants. Nine test markers were assayed including IL-6, IL-I 0, ILI2, TNF-α, lipopolysaccharide binding protein, procalcitonin, sE-selectin, sICAM -1 and a potential differential marker for Gram-positive sepsis- anti-lipid S antibody. A total of 445 patients were enrolled into this study from the Queen Elizabeth Hospital and Selly Oak Hospital (Birmingham). The results showed that all the markers were elevated in patients with sepsis and that patients with a Gram-negative sepsis consistently produced higher median/range serum levels than those with a Gram-positive sepsis. No single marker was able to identify all the septic patients. Combining two markers caused the sensitivities and specificities for a diagnosis of sepsis to increase to within a 90% to 100% range. By a process of elimination the markers that survived into the last phase were IL-6 with sICAM -1, and anti-lipid S IgG assays Defining cut-off levels for a diagnosis of sepsis became problematic and a semi-blind trial was devised to test the markers in the absence of both clinical details and positive blood cultures. Patients with pyrexia of unknown origin and negative BC were included in this phase (4). The results showed that IL-6 with sICAM-l are authentic markers of sepsis. There was 82% agreement between the test marker diagnosis and the clinical diagnosis for sepsis in patients with a Gram-positive BC and 78% agreement in cases of Gram-negative Be. In the PUO group the test markers identified 12 cases of sepsis and the clinical diagnosis 15. The markers were shown to differentiate between early sepsis and sepsis, inflammatory responses and infection. Anti-lipid S with IL-6 proved be a sensitive marker for Gram-positive infections/sepsis.

Age-related macular degeneration and modification of systemic complement factor H production through liver transplantation

Purpose: To investigate whether modification of liver complement factor H (CFH) production, by alteration of liver CFH Y402H genotype through liver transplantation (LT), influences the development of age-related macular degeneration (AMD). Design: Multicenter, cross-sectional study. Participants: We recruited 223 Western European patients ≥55 years old who had undergone LT ≥5 years previously. Methods: We determined AMD status using a standard grading system. Recipient CFH Y402H genotype was obtained from DNA extracted from recipient blood samples. Donor CFH Y402H genotype was inferred from recipient plasma CFH Y402H protein allotype, measured using enzyme-linked immunosorbent assays. This approach was verified by genotyping donor tissue from a subgroup of patients. Systemic complement activity was ascertained by measuring levels of plasma complement proteins using an enzyme-linked immunosorbent assay, including substrates (C3, C4), activation products (C3a, C4a, and terminal complement complex), and regulators (total CFH, C1 inhibitor). Main Outcome Measures: We evaluated AMD status and recipient and donor CFH Y402H genotype. Results: In LT patients, AMD was associated with recipient CFH Y402H genotype (P = 0.036; odds ratio [OR], 1.6; 95% confidence interval [CI], 1.0-2.4) but not with donor CFH Y402H genotype (P = 0.626), after controlling for age, sex, smoking status, and body mass index. Recipient plasma CFH Y402H protein allotype predicted donor CFH Y402H genotype with 100% accuracy (n = 49). Plasma complement protein or activation product levels were similar in LT patients with and without AMD. Compared with previously reported prevalence figures (Rotterdam Study), LT patients demonstrated a high prevalence of both AMD (64.6% vs 37.1%; OR, 3.09; P<0.001) and the CFH Y402H sequence variation (41.9% vs 36.2%; OR, 1.27; P = 0.014). Conclusions: Presence of AMD is not associated with modification of hepatic CFH production. In addition, AMD is not associated with systemic complement activity in LT patients. These findings suggest that local intraocular complement activity is of greater importance in AMD pathogenesis. The high AMD prevalence observed in LT patients may be associated with the increased frequency of the CFH Y402H sequence variation. © 2013 by the American Academy of Ophthalmology Published by Elsevier Inc.

Monocyte urokinase-type plasminogen activator up-regulation reduces thrombus size in a model of venous thrombosis

Background - Our previous studies showed that the direct injection of an adenovirus construct expressing urokinase-type plasminogen activator (uPA) into experimental venous thrombi significantly reduces thrombus weight. The systemic use of adenovirus vectors is limited by inherent hepatic tropism and inflammatory response. As macrophages are recruited into venous thrombi, it is reasonable to speculate that these cells could be used to target the adenovirus uPA (ad-uPA) gene construct to the thrombus. The aims of this study were to determine whether macrophages transduced with ad-uPA have increased fibrinolytic activity and whether systemic injection of transduced cells could be used to target uPA expression to the thrombus and reduce its size. Methods - The effect of up-regulating uPA was examined in an immortalized macrophage cell line (MM6) and macrophages differentiated from human blood monocyte-derived macrophages (HBMMs). Cells were infected with ad-uPA or blank control virus (ad-blank). Fibrinolytic mediator expression, cell viability, and cytokine expression were measured by activity assays and enzyme-linked immunosorbent assays. Monocyte migration was measured using a modified Boyden chamber assay. A model of venous thrombosis was developed and characterized in mice with severe combined immunodeficiency (SCID). This model was used to study whether systemically administered macrophages over-expressing uPA reduced thrombus size. Uptake of HBMMs into the thrombus induced in these mice was confirmed by a combination of PKH2-labeled cell tracking and colocalization with human leukocyte antigen (HLA) by immunohistology. Results - Compared with ad-blank, treated HBMMs transduction with ad-uPA increased uPA production by >1000-fold (P = .003), uPA activity by 150-fold (P = .0001), and soluble uPA receptor (uPAR) by almost twofold (P = .043). Expression of plasminogen activator inhibitor (PAI-1) and PAI-2 was decreased by about twofold (P = .011) and threefold (P = .005), respectively. Up-regulation of uPA had no effect on cell viability or inflammatory cytokine production compared with ad-blank or untreated cells. Ad-uPA transduction increased the migration rate of HBMMs (about 20%, P = .03) and MM6 cells (>twofold, P = .005) compared with ad-blank treated controls. Human macrophage recruitment into the mouse thrombus was confirmed by the colocalization of HLA with the PKH2-marked cells. Systemic injection of uPA-up-regulated HBMMs reduced thrombus weight by approximately 20% compared with ad-blank (P = .038) or sham-treated controls (P = .0028). Conclusion - Transduction of HBBM with ad-uPA increases their fibrinolytic activity. Systemic administration of uPA up-regulated HBBMs reduced thrombus size in an experimental model of venous thrombosis. Alternative methods of delivering fibrinolytic agents are worth exploring.

The serological diagnosis of staphylococcal infective endocarditis

Objectives: Establishing the diagnosis of infective endocarditis (IE) can be difficult when blood cultures remain sterile or echocardiography is inconclusive. Staphylococcus aureus is a common aetiological microorganism in IE and is associated with severe valvular destruction and increased mortality. Early diagnosis using culture and antibiotic independent tests would be preferable to allow prompt antibiotic administration. We have developed and evaluated 2 serological assays for the rapid identification of a staphylococcal aetiology in infective endocarditis. The assays measure IgG against whole cells of S. aureus and IgG against lipid S, a novel extracellular antigen released by Gram-positive microorganisms. Methods: Serum was collected from 130 patients with IE and 94 control patients. IgG against whole cells of S. aureus and against lipid S was measured by enzyme linked immunosorbent assay (ELISA). Results: Anti-lipid S IgG titres were higher in IE caused by Gram-positive microorganisms than in controls (p < 0.0001) and higher in staphylococcal IE than in both controls and IE caused by other microorganisms (p = 0.0003). Anti-whole cell staphylococcal IgG was significantly higher in serum from patients with staphylococcal IE than in IE caused by other microorganisms and control samples (p < 0.0001). Conclusion: High anti-whole cell IgG titres are predictive of a staphylococcal aetiology in IE. Elevated serum anti-lipid S IgG titres are predictive of Gram-positive infection compared to controls, very high titres being associated with staphylococcal IE. © 2005 The British Infection Society.

Rotational co-culture of clonal β-cells with endothelial cells:effect of PPAR-γ agonism in vitro on insulin and VEGF secretion

Aim: Delayed graft revascularization impedes the success of human islet transplantation. This study utilized rotational co-culture of insulin secreting ß-cells with human umbilical vein endothelial cells (HUVECs) and a peroxisome proliferator-activated receptor gamma (PPAR-?) agonist to promote insulin and vascular endothelial growth factor (VEGF) secretory function. Methods: Clonal BRIN-BD11 (D11) cells were maintained in static culture (SC) and rotational culture (RC) ± HUVEC and ± the TZD (thiazolidinedione) rosiglitazone (10 mmol/l) as a specific PPAR-? agonist. HUVECs were cultured in SC and RC ± D11 and ± TZD. D11 insulin secretion was induced by static incubation with low glucose (1.67 mmol/l), high glucose (16.7 mmol/l) and high glucose with 10 mmol/l theophylline (G+T) and assessed by enzyme-linked immunosorbent assay (ELISA). HUVEC proliferation was determined by ATP luminescence, whereas VEGF secretion was quantified by ELISA. Co-cultured cells were characterized by immunostaining for insulin and CD31. Results: D11 SC and RC showed enhanced insulin secretion in response to 16.7 mmol/l and G+T (p <0.01); without significant alteration by the TZD. Co-culture with HUVEC in SC and RC also increased D11 insulin secretion when challenged with 16.7 mmol/l and G+T (p <0.01), and this was slightly enhanced by the TZD. The presence of HUVEC increased D11 SC and RC insulin secretion in response to high glucose and G+T, respectively (p <0.01). Addition of the TZD increased SC and RC HUVEC ATP content (p <0.01) and VEGF production (p <0.01) in the presence and absence of D11 cells. Conclusions: Rotational co-culture of insulin secreting cells with endothelial cells, and exposure to a PPAR-? agonist may improve the prospects for graft revascularization and function after implantation. © 2011 Blackwell Publishing Ltd.

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.

Investigating ocular allergy:can we determine a better measurement?

Objective: Ocular allergy is a broad group of allergic conditions involving inflammation of the conjunctiva and the most common forms are seasonal allergic conjunctivitis (SAC; 90% of cases) and perennial allergic conjunctivitis (PAC; 5% of cases). The main symptom is ocular itching caused by mast cell degranulation leading to the release of histamine and other mediators such as tryptase. Tryptase is a neutral protease that is selectively concentrated in the secretory granules of human mast cells and has been shown to be a sensitive and specific marker of type I hypersensitivity reaction. The objective was to ascertain the best assay method for determining the tryptase levels in tear samples and whether this can be used to determine the efficacy of non-pharmacological treatments compared to no treatment or their combined effect with anti-allergic medication for SAC and PAC. Method: Thirty patients with a history of SAC were recruited into a randomised blind study during winter months when all the patients were asymptomatic. Suitability was determined by skin prick and conjunctival provocation tests. Patients were randomly assigned to either a non-pharmacological or a pharmacological Intervention group and received each test condition assigned to their group in a randomly assigned order. Symptoms were provoked by exposure to pollen in an environmental test chamber where the temperature, humidity and grass pollen levels were set to a high pollen count day. Tear samples were taken set intervals during the visit and then processed by enzyme linked immunosorbent assay (ELISA) for the detection of tryptase levels. Preliminary results: Results are still being analysed but the preliminary optimisation experiments tested four different ELISA systems; two indirect assays and two capture ‹sandwich› assays. The results suggest that in both sandwich assay systems non-specific binding occurred which could not be easily overcome. The indirect assay systems both showed specific reactions, and the sensitivity achieved was greater with the monoclonal than the polyclonal antibody. Using these findings the indirect assay system was optimised to provide a standardised system for measuring tryptase. Initial trials using human tear samples displayed tryptase levels between 23.1 and 175.1 ng/ml; levels which fall within the anticipated range for patients with SAC. Further statistical work is needed to determine whether tryptase levels vary between the treatments 75.

Immunochemical detection of UV-induced DNA damage and repair

The application of an antiserum to ultraviolet radiation (UVR)-damaged DNA is presented. A novel experimental system was employed to ascertain the limits of detection for this antiserum. Using a DNA standard containing a known amount of dimer, the limits of detection were found to be 0.9 fmol of dimer. This was compared to a limit of 20-50 fmol dimer using gas chromatography-mass spectrometry (GC-MS). Induction of thymine dimers in DNA following UVR exposure, as assessed using this antiserum in an enzyme-linked immunosorbent assay (ELISA), was compared with GC-MS measurements. The ELISA method successfully demonstrated the induction of lesions in DNA irradiated either with UVC or UVB, although despite high sensitivity, no discernible binding was seen to UVA-irradiated DNA. The antiserum was also shown to be applicable to immunocytochemistry, localising damage in the nuclei of UVR exposed keratinocytes in culture. The ability of the antiserum to detect DNA damage in skin biopsies of individuals exposed to sub-erythemal doses of UVR was also demonstrated. Moreover, the subsequent removal of this damage, as evidenced by a reduction in antiserum staining, was noted in sections of biopsies taken in the hours following irradiation. © 2003 Elsevier B.V. All rights reserved.

Serodiagnosis of staphylococcal sepsis

The coagulase-negative staphylococci are the most frequent cause of sepsis associated with indwelling intravascular catheters. Current microbiological investigations to support the diagnosis of catheter-related sepsis (CRS) include the culture of blood and catheter tips, however positive results may reflect specimen contamination, or colonisation of the catheter rather than true sepsis. Previous serological approaches to assist in the diagnosis of CRS based on cellular staphylococcal antigens have been of limited value. In this current study, the serodiagnostic potential of an exocellular antigen produced by 7 strains of coagulase-negative staphylococci cultured in brain heart infusion broth was investigated. Antigenic material isolated by gel permeation from liquid culture was characterised by immunological techniques and chemical analysis. Characterisation of the exocellular antigen revealed a novel glycerophosphoglycolipid, termed lipid S. which shared antigenic determinants with lipoteichoic acid, but differed by comprising a glycerophosphate chain length of only 6 units. In addition, lipid S was immunologically distinct from diphosphatidyl glycerol, a constituent cell membrane phospho lipid. An indirect enzyme linked immunosorbent assay (ELISA) based on lipid S was subsequently developed and used to determine serum antibody levels (IgM and IgG) in 67 patients with CRS due to staphylococci, and 67 patients with a central venous catheter (CVC) in situ who exhibited no evidence of sepsis. The sensitivity and specificity of the lipid S IgG ELISA was 75% and 90% respectively whilst the IgM assay had sensitivity and specificity of 52% and 85%. The addition of GullSORereagent to the EL1SA procedure to remove competing serum IgG and rheumatoid factor did not significantly improve the performance of the IgM assay. The serological response in serial serum samples of 13 patients with CRS due to staphylococci was investigated. Elevated levels of antibody were detected at an early stage of infection, prior to the isolation of microorganisms by standard culture methods, and before the clinical presentation of sepsis in 3 patients. The lipid S ELISA was later optimised and a rapid 4-hour assay developed for the serodiagnosis of CRS. Serum IgG levels were determined in 40 patients with CRS due to staphylococci and 40 patients with a CVC in situ who exhibited no evidence of sepsis. The sensitivity and specificity of the rapid IgG assay was 70% and 100% respectively. Elevated serum antibody levels in patients with endocarditis, prosthetic joint infection and pyogenic spondylodiscitis due to Gram-positive cocci were also detected with the lipid S ELISA suggesting that the assay may facilitate the diagnosis of these infections. Unexpected increased levels of anti-lipid S IgG in 31% of control patients with sciatica suggested a possible microbial aetiology of this condition. Further investigation of some of these patients by culture of microdiscectomy tissue removed at operation, revealed the presence of low-virulent microorganisms in 37% of patients of which Propionibacterium aeries accounted for 85% of the positive culture isolates. The results suggested a previously unrecognised association between P. acnes and sciatica, which may have implications for the future management of the condition.

Influence of iron on bacterial infections in leukaemia

The influence of iron metabolism, both on the invading bacterial pathogen and in the host is widespread and often appears to be crucial in determining the outcome of an infection. This study involved the investigation of leukaemia, a clinical disease where abnormal availability of iron may play a part in predisposing patients to bacterial infection. The iron status throughout a Gram-negative septicaemia and in 20 random, newly diagnosed leukaemic patients was assessed. The results revealed that the majority of the patients exhibited high serum iron levels and serum transferrin saturation often at 100%, with an inability to reduce the latter to within normal values during an infection episode. The antibody response to P.aeruginosa, E.coli and K.pneumoniae outer membrane protein (OMP) antigens were investigated by immunoblotting with sequential serum samples during infection in the leukaemic host. Antibodies to all the major OMPs, were observed, although recognition of iron-regulated membrane proteins (IRMPs) was in many cases weak. Results from the enzyme-linked immunosorbent assay indicated that in all patients antibody titre in response to infection was poor. Sub-MICs of mitomycin C significantly altered the surface characteristics of P.aeruginosa. The silver-stained SDS-PAGE gels of proteinase K digested whole cell lysates of strains PAO1, 6750, M7 and PAJ indicated that core LPS was affected in the presence of mitomycin C. In contrast, the rough strain AK1012 showed no observable differences. Results obtained using quantitative gas-liquid chromatographic analysis showed the amount of LPS fatty acids to be unaffected, however, the KDO and carbohydrate content in strains PAO1, 6750 and M7 under Fe+ and Fe- growth conditions were decreased by up to 4-fold in the presence of mitomycin C, indicating perturbed expression of LPS. The cell surface became significantly more hydrophobic in the P.aeruginosa strains, except AK1012 which was comparatively unaffected. The induction of protein G (OprG) in P.aeruginosa was found to be a sensitive indicator of media iron. The data indicated that expression of OprG can be modulated by growth rate/phase, availability of iron and by the presence of ciprofloxacin in the growth medium.

Oxygen tension modulates the cytokine response of oral epithelium to periodontal bacteria

Background: There is an inverse relationship between pocket depth and pocket oxygen tension with deep pockets being associated with anaerobic bacteria. However, little is known about how the host tissues respond to bacteria under differing oxygen tensions within the periodontal pocket. Aim: To investigate the effect of different oxygen tensions upon nuclear factor-kappa B (NF-?B) activation and the inflammatory cytokine response of oral epithelial cells when exposed to nine species of oral bacteria. Materials and Methods: H400 oral epithelial cells were equilibrated at 2%, 10% or 21% oxygen. Cells were stimulated with heat-killed oral bacteria at multiplicity of infection 10:1, Escherichia coli lipopolysaccharide (15 µg/ml) or vehicle control. Interleukin-8 (IL-8) and tumour necrosis factor-alpha (TNF-a) levels were measured by enzyme-linked immunosorbent assay and NF-?B activation was measured by reporter vector or by immunohistochemical analysis. Results: Tannerella forsythensis, Porphyromonas gingivalis and Prevotella intermedia elicited the greatest epithelial NF-?B activation and cytokine responses. An oxygen-tension-dependent trend in cytokine production was observed with the highest IL-8 and TNF-a production observed at 2% oxygen and lowest at 21% oxygen. Conclusions: These data demonstrate a greater pro-inflammatory host response and cell signalling response to bacteria present in more anaerobic conditions, and hypersensitivity of epithelial cells to pro-inflammatory stimuli at 2% oxygen, which may have implications for disease pathogenesis and/or therapy.

Dysregulation of hydrogen sulfide producing enzyme cystathionine γ-lyase contributes to maternal hypertension and placental abnormalities in preeclampsia

Background—The exact etiology of preeclampsia is unknown, but there is growing evidence of an imbalance in angiogenic growth factors and abnormal placentation. Hydrogen sulfide (H2S), a gaseous messenger produced mainly by cystathionine ?-lyase (CSE), is a proangiogenic vasodilator. We hypothesized that a reduction in CSE activity may alter the angiogenic balance in pregnancy and induce abnormal placentation and maternal hypertension. Methods and Results—Plasma levels of H2S were significantly decreased in women with preeclampsia (P<0.01), which was associated with reduced placental CSE expression as determined by real-time polymerase chain reaction and immunohistochemistry. Inhibition of CSE activity by DL-propargylglycine reduced placental growth factorproduction from first-trimester (8–12 weeks gestation) human placental explants and inhibited trophoblast invasion in vitro. Knockdown of CSE in human umbilical vein endothelial cells by small-interfering RNA increased the release of soluble fms-like tyrosine kinase-1 and soluble endoglin, as assessed by enzyme-linked immunosorbent assay, whereas adenoviral-mediated CSE overexpression in human umbilical vein endothelial cells inhibited their release. Administration of DL-propargylglycine to pregnant mice induced hypertension and liver damage, promoted abnormal labyrinth vascularization in the placenta, and decreased fetal growth. Finally, a slow-releasing H2S-generating compound, GYY4137, inhibited circulating soluble fms-like tyrosine kinase-1 and soluble endoglin levels and restored fetal growth in mice that was compromised by DL-propargylglycine treatment, demonstrating that the effect of CSE inhibitor was attributable to inhibition of H2S production. Conclusions—These results imply that endogenous H2S is required for healthy placental vasculature and that a decrease in CSE/H2S activity may contribute to the pathogenesis of preeclampsia. (Circulation. 2013;127:2514-2522.)

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).

Dysregulation of hydrogen sulfide producing enzyme cystathionine γ-lyase contributes to maternal hypertension and placental abnormalities in preeclampsia

Background-The exact etiology of preeclampsia is unknown, but there is growing evidence of an imbalance in angiogenic growth factors and abnormal placentation. Hydrogen sulfide (H2S), a gaseous messenger produced mainly by cystathionine γ-lyase (CSE), is a proangiogenic vasodilator. We hypothesized that a reduction in CSE activity may alter the angiogenic balance in pregnancy and induce abnormal placentation and maternal hypertension. Methods and Results-Plasma levels of H2S were significantly decreased in women with preeclampsia (P<0.01), which was associated with reduced placental CSE expression as determined by real-time polymerase chain reaction and immunohistochemistry. Inhibition of CSE activity by DL-propargylglycine reduced placental growth factorproduction from first-trimester (8-12 weeks gestation) human placental explants and inhibited trophoblast invasion in vitro. Knockdown of CSE in human umbilical vein endothelial cells by small-interfering RNA increased the release of soluble fms-like tyrosine kinase-1 and soluble endoglin, as assessed by enzyme-linked immunosorbent assay, whereas adenoviral-mediated CSE overexpression in human umbilical vein endothelial cells inhibited their release. Administration of DL-propargylglycine to pregnant mice induced hypertension and liver damage, promoted abnormal labyrinth vascularization in the placenta, and decreased fetal growth. Finally, a slow-releasing H2S-generating compound, GYY4137, inhibited circulating soluble fms-like tyrosine kinase-1 and soluble endoglin levels and restored fetal growth in mice that was compromised by DL-propargylglycine treatment, demonstrating that the effect of CSE inhibitor was attributable to inhibition of H2S production. Conclusions-These results imply that endogenous H2S is required for healthy placental vasculature and that a decrease in CSE/H2S activity may contribute to the pathogenesis of preeclampsia. © 2013 American Heart Association, Inc.