Maternal serum glycosylated fibronectin as a point-of-care biomarker for assessment of preeclampsia.

OBJECTIVE: We assessed the association of glycosylated fibronectin (GlyFn) with preeclampsia and its performance in a point-of-care (POC) test. STUDY DESIGN: GlyFn, placental growth factor (PlGF), and soluble vascular endothelial growth factor receptor 1 (sFlt1) levels were determined in serum samples from 107 pregnant women. In all, 45 were normotensive and 62 were diagnosed with preeclampsia. The ability of GlyFn to assess preeclampsia status and relationships between GlyFn and maternal characteristics and pregnancy outcomes were analyzed. RESULTS: GlyFn serum levels in the first trimester were significantly higher in women with preeclampsia (P < .01) and remained higher throughout pregnancy (P < .01). GlyFn, sFlt1, PlGF, and the sFlt1/PlGF ratio were significantly associated (P < .01) with preeclampsia status, and the classification performance of these analytes represented by area under the receiver operating characteristic curve was 0.99, 0.96, 0.94, and 0.98, respectively, with 95% confidence intervals of 0.98-1.00, 0.89-1.00, 0.86-1.00, and 0.94-1.00, respectively. Increased GlyFn levels were significantly associated with gestational age at delivery (P < .01), blood pressure (P = .04), and small-for-gestational-age neonates. Repeated-measures analysis of the difference in weekly GlyFn change in the third trimester demonstrated that mild preeclampsia was associated with a weekly change of 81.7 µg/mL (SE 94.1) vs 195.2 µg/mL (SE 88.2) for severe preeclampsia. The GlyFn POC demonstrated similar performance to a plate assay with an area under the receiver operating characteristic curve of 0.93 and 95% confidence interval of 0.85-1.00. CONCLUSION: GlyFn is a robust biomarker for monitoring of preeclampsia in both a standard and POC format, which supports its utility in diverse settings.

Roles of the sodium-translocating NADH:quinone oxidoreductase (Na+-NQR) on vibrio cholerae metabolism, motility and osmotic stress resistance.

The Na+ translocating NADH:quinone oxidoreductase (Na+-NQR) is a unique respiratory enzyme catalyzing the electron transfer from NADH to quinone coupled with the translocation of sodium ions across the membrane. Typically, Vibrio spp., including Vibrio cholerae, have this enzyme but lack the proton-pumping NADH:ubiquinone oxidoreductase (Complex I). Thus, Na+-NQR should significantly contribute to multiple aspects of V. cholerae physiology; however, no detailed characterization of this aspect has been reported so far. In this study, we broadly investigated the effects of loss of Na+-NQR on V. cholerae physiology by using Phenotype Microarray (Biolog), transcriptome and metabolomics analyses. We found that the V. cholerae ΔnqrA-F mutant showed multiple defects in metabolism detected by Phenotype Microarray. Transcriptome analysis revealed that the V. cholerae ΔnqrA-F mutant up-regulates 31 genes and down-regulates 55 genes in both early and mid-growth phases. The most up-regulated genes included the cadA and cadB genes, encoding a lysine decarboxylase and a lysine/cadaverine antiporter, respectively. Increased CadAB activity was further suggested by the metabolomics analysis. The down-regulated genes include sialic acid catabolism genes. Metabolomic analysis also suggested increased reductive pathway of TCA cycle and decreased purine metabolism in the V. cholerae ΔnqrA-F mutant. Lack of Na+-NQR did not affect any of the Na+ pumping-related phenotypes of V. cholerae suggesting that other secondary Na+ pump(s) can compensate for Na+ pumping activity of Na+-NQR. Overall, our study provides important insights into the contribution of Na+-NQR to V. cholerae physiology.

NhaP1 is a K+(Na+)/H+ antiporter required for growth and internal pH homeostasis of Vibrio cholerae at low extracellular pH.

Vibrio cholerae has adapted to a wide range of salinity, pH and osmotic conditions, enabling it to survive passage through the host and persist in the environment. Among the many proteins responsible for bacterial survival under these diverse conditions, we have identified Vc-NhaP1 as a K(+)(Na(+))/H(+) antiporter essential for V. cholerae growth at low environmental pH. Deletion of the V. cholerae nhaP1 gene caused growth inhibition when external potassium was either limited (100 mM and below) or in excess (400 mM and above). This growth defect was most apparent at mid-exponential phase, after 4-6 h of culture. Using a pH-sensitive GFP, cytosolic pH was shown to be dependent on K(+) in acidic external conditions in a Vc-NhaP1-dependent manner. When functionally expressed in an antiporterless Escherichia coli strain and assayed in everted membrane vesicles, Vc-NhaP1 operated as an electroneutral alkali cation/proton antiporter, exchanging K(+) or Na(+) ions for H(+) within a broad pH range (7.25-9.0). These data establish the putative V. cholerae NhaP1 protein as a functional K(+)(Na(+))/H(+) antiporter of the CPA1 family that is required for bacterial pH homeostasis and growth in an acidic environment.

The putative Na+/H+ antiporter of Vibrio cholerae, Vc-NhaP2, mediates the specific K+/H+ exchange in vivo.

The existence of bacterial K(+)/H(+) antiporters that prevent the overaccumulation of potassium in the cytoplasm was predicted by Peter Mitchell almost 50 years ago. The importance of K(+)/H(+) antiport for bacterial physiology is widely recognized, but its molecular mechanisms remain underinvestigated. Here, we demonstrate that a putative Na(+)/H(+) antiporter, Vc-NhaP2, protects cells of Vibrio cholerae growing at pH 6.0 from high concentrations of external K(+). Resistance of V. cholerae to Na(+) was found to be independent of Vc-NhaP2. When assayed in inside-out membrane vesicles derived from antiporter-deficient Escherichia coli, Vc-NhaP2 catalyzed the electroneutral K(+)(Rb(+))/H(+) exchange with a pH optimum of approximately 7.75 with an apparent K(m) for K(+) of 1.62 mM. In the absence of K(+), it exhibited Na(+)/H(+) antiport, albeit rather weakly. Interestingly, while Vc-NhaP2 cannot exchange Li(+) for protons, elimination of functional Vc-NhaP2 resulted in a significantly higher Li(+) resistance of V. cholerae cells growing at pH 6.0, suggesting the possibility of Vc-NhaP2-mediated Li(+)/K(+) antiport. The peculiar cation specificity of Vc-NhaP2 and the presence of its two additional paralogues in the same genome make this transporter an attractive model for detailed analysis of the structural determinants of the substrate specificity in alkali cation exchangers.

Elevation of transforming growth factor beta (TGFbeta) and its downstream mediators in subcutaneous foreign body capsule tissue.

Foreign body encapsulation represents a chronic fibrotic response and has been a major obstacle that reduces the useful life of implanted biomedical devices. The precise mechanism underlying such an encapsulation is still unknown. We hypothesized that, considering its central role in many other fibrotic conditions, transforming growth factor beta (TGFbeta) may play an important role during the formation of foreign body capsule (FBC). In the present study, we implanted mock sensors in rats subcutaneously and excised FBC samples at day 7, 21, and 48-55 postimplantation. The most abundant TGFbeta isoform in all tissues was TGFbeta1, which was expressed minimally in control tissue. The expression of both TGFbeta1 RNA and protein was significantly increased in FBC tissues at all time points, with the highest level in day 7 FBC. The number of cells stained for phosphorylated Smad2, an indication of activated TGFbeta signaling, paralleled the expression of TGFbeta. A similar dynamic change was also observed in the numbers of FBC myofibroblasts, which in response to TGFbeta, differentiate from quiescent fibroblasts and synthesize collagen. Type I collagen, the most prominent downstream target of TGFbeta in fibrosis, was found in abundance in the FBC, especially during the latter time periods. We suggest that TGFbeta plays an important role in the FBC formation. Inhibition of TGFbeta signaling could be a promising strategy in the prevention of FBC formation, thereby extending the useful life of subcutaneous implants.

Passivating protein coatings for implantable glucose sensors: evaluation of protein retention.

The long-term function of implantable biosensors is limited by the foreign-body reaction (FBR). Since the acute phase of the FBR involves macrophage attachment mediated by adsorbed fibrinogen, preadsorption, and retention of other proteins might reduce the FBR. The retention of preadsorbed albumin, hemoglobin, von Willebrand's factor, and high-molecular-weight kininogen was therefore measured after exposure to plasma. The retention of preadsorbed proteins after incubation with monocyte cultures and implantation in rats was also measured. Fibrinogen adsorption from plasma to the preadsorbed surfaces was also measured. Hemoglobin adsorption was higher than that for other proteins, and it also had the greatest retention after exposure to blood plasma. When surfaces preadsorbed with hemoglobin were incubated with monocytes, more of the hemoglobin was displaced than that after incubation in plasma, while still more hemoglobin was displaced when the surfaces were implanted in vivo. Protein preadsorption on polystyrene greatly reduced fibrinogen adsorption. However, polyurethane surfaces used for glucose sensors had low fibrinogen adsorption compared with polystyrene, and this low level was not further reduced by preadsorption with other proteins. Preadsorbed proteins on polymers appear to be removed by passive exchange and/or displacement by plasma proteins and by proteases released by monocytes.

A novel insulin delivery algorithm in rats with type 1 diabetes: the fading memory proportional-derivative method.

An algorithm designed to automatically control insulin delivery was tested in rats with Type 1 diabetes. This nonlinear algorithm included a fading memory component of proportional and derivative errors in order to simulate normal insulin secretion. Error-weighting functions for the proportional and derivative terms were used with a performance index designed for error adaptation. In the first version of the algorithm, the proportional gain was adaptively varied. In the second version, a low rate of basal insulin delivery was adaptively varied. Six 6-h studies with each version were conducted using frequent blood sampling and intravenous insulin delivery. In Version 2 studies, blood glucose levels during the last two hours were well-controlled and significantly lower than in Version 1 (118 +/- 2.0 vs. 130 +/- 2.9 mg/dL). Neither version produced hypoglycemia. Future research using this algorithm needs to focus on automated glucose sensing in combination with insulin delivery.

An implantable subcutaneous glucose sensor array in ketosis-prone rats: closed loop glycemic control.

A closed loop system of diabetes control would minimize hyperglycemia and hypoglycemia. We therefore implanted and tested a subcutaneous amperometric glucose sensor array in alloxan-diabetic rats. Each array employed four sensing units, the outputs of which were processed in real time to yield a unified signal. We utilized a gain-scheduled insulin control algorithm which rapidly reduced insulin delivery as glucose concentration declined. Such a system was generally effective in controlling glycemia and the degree of lag between blood glucose and the sensor signal was usually 3-8 min. After prolonged implantation, this lag was sometimes longer, which led to impairment of sensor accuracy. Using a prospective two-point calibration method, sensor accuracy and closed loop control were good. A revised algorithm yielded better glycemic control than the initial algorithm did. Future research needs to further improve calibration methods and reduce foreign body fibrosis in order to avoid a time-related increase in lag duration.

Induction of type-1 diabetes mellitus in laboratory rats by use of alloxan: route of administration, pitfalls, and insulin treatment.

Uncertainties have existed regarding the systematic induction and management of drug-induced diabetes mellitus (DM). Issues have included the optimal route of administration of the drug, methods of reducing drug toxicosis and mortality, how to induce type-1 versus type-2 DM, and how to manage labile DM in rats. In attempting to induce type-1 DM in Sprague-Dawley rats, we classified hyperglycemic animals as having type-1 DM only if their post-treatment blood ketone concentration was high. We found that multiple doses of alloxan led to significantly higher mortality than did a single dose. A single high dose (200 mg/kg of body weight given intraperitoneally) was the best treatment and led to 70% incidence of type-1 DM and only 10% mortality. In contrast, intravenous administration of similar doses was toxic. Assiduous management of alloxan-induced DM is crucial to avoid severe hypoglycemia from massive insulin release and to avoid diabetic ketoacidosis. Frequent glucose monitoring and appropriate administration of carbohydrate and fluids is necessary during this stage. For long-term management, daily administration of long-acting insulin (glargine) appears to be safe and effective. Rapid-acting insulins reduce glucose concentration rapidly, and must be used with caution. If specific precautions are observed, intraperitoneal administration of high-dose alloxan to laboratory rats leads to a condition that closely resembles human type-1 DM.

The effect of local subcutaneous delivery of vascular endothelial growth factor on the function of a chronically implanted amperometric glucose sensor.

The foreign body capsule that forms around implanted devices such as glucose sensors is hypovascular and has limited permeability to glucose. Such a capsule may function better if well vascularized. We hypothesized that capsular vascularization achieved by local release of vascular endothelial growth factor (VEGF) would lead to enhanced function. Amperometric glucose sensor array disks, each with four indicating electrodes, were implanted into rats. Animals received local subcutaneous infusions of VEGF(165) via osmotic pumps at a location on the sensor face 2 mm from one of the electrodes ("near units"). "Intermediate" electrode units were 15 mm, and "distant" units were 22 mm, from the VEGF source. Every 2 weeks, a glucose infusion was given to assess sensor function by telemetry. Near units demonstrated a lower lag duration (delay after blood glucose) than intermediate and distant units. The mean absolute relative difference for near units was less than for distant units. The percentage of data pairs in the A region of the Clarke error grid of the near sensing units was greater than that of the distant units. Values for the functional measures for saline controls fell between near and distant VEGF values. Glucose sensor function was found to be more favorable in units immediately adjacent to the VEGF infusion port. The most likely cause for this finding is increased neovessel growth in the surrounding foreign body capsule. Slow release of angiogenic growth factors may be a potential method for chronically enhancing the function of a subcutaneously implanted biosensor.

Vascularizing the tissue surrounding a model biosensor: how localized is the effect of a subcutaneous infusion of vascular endothelial growth factor (VEGF)?

Implantable continuous biosensors would improve disease management but long term function of such devices have been limited by a hypovascular foreign body capsule that inhibits influx of analytes. To assess whether capsule vascularity could be increased, we studied the histologic effects of a 28-day continuous infusion of vascular endothelial growth factor (VEGF) (0.45 microg/day) vs. saline from the surface of a model disk biosensor that was implanted subcutaneously in rats. At day 40, tissue was obtained at varying distances from the infusion port and capsular microvessels were counted using two histologic techniques. VEGF treatment led to a marked increase in capillary density. In tissue located 1 mm away from the infusion site, capillary density in VEGF-treated animals was 200-300% higher than in saline controls. Tissue located 13 mm away, but not 25 mm away, also demonstrated neovascularization. Serum obtained from a distant vein during the infusion did not show an elevated concentration of VEGF. These data demonstrate that a subcutaneous infusion of VEGF creates localized neovascularization of the foreign body capsule and suggest that systemic effects of VEGF are avoidable. Vascularization of a foreign body capsule surrounding a subcutaneous biosensor might well extend its useful life.

A fully implantable subcutaneous glucose sensor array: enhanced accuracy from multiple sensing units and a median-based algorithm.

Although continuous electrochemical glucose monitoring holds promise in the management of diabetes, its utility is limited in part because of error of unclear origin. The use of redundant glucose sensors in an array might reduce such error. We hypothesized that in a subcutaneously implanted array, a median-based continuous computation that excludes outlying data would lead to more accurate glucose measurement than averaging of all signals. Each rat was implanted with an array of four sensing units, and each unit transmitted data independently to an external monitoring device. Animals underwent perturbation of glucose by insulin infusions in diabetic animals and glucose infusions in nondiabetic animals, and in both, capillary glucose monitoring was performed frequently. Repeat glucose perturbation studies were performed every 1-2 weeks. We observed that a median-based technique, the Z-score with Median Absolute Deviation (ZMAD), consistently led to greater sensing accuracy as compared with signal averaging. The ZMAD technique yielded a correlation coefficient of 0.93, and 96% of values fell in the A and B regions of the Clarke error grid, demonstrating a high degree of accuracy of the unified signal. When tested in an implanted array of glucose sensors, a median-based technique (ZMAD) yields an accurate unified signal, and its accuracy is superior to signal averaging.