Challenges and solutions in the development of genomic biomarker panels: a systematic phased approach.

In the post-genome era, high throughput gene expression profiling has been successfully used to develop genomic biomarker panels (GBP) that can be integrated into clinical decision making. The development of GBPs in the context of personalized medicine is a scientifically challenging and resource-intense process. It needs to be accomplished in a systematic phased approach to address biological variation related to a clinical phenotype (e.g. disease etiology, gender, etc.) and minimize technical variation (noise). Here we present the methodological aspects of GBP development based on the experience of the Cardiac Allograft Rejection Gene Expression Observation (CARGO) study, a study that lead to the development of a molecular classifier for rejection screening in heart transplant patients.

Vascular endothelial growth factor and dexamethasone release from nonfouling sensor coatings affect the foreign body response.

Vascular endothelial growth factor (VEGF) and dexamethasone (DX) release from hydrogel coatings were examined as a means to modify tissue inflammation and induce angiogenesis. Antibiofouling hydrogels for implantable glucose sensor coatings were prepared from 2-hydroxyethyl methacrylate, N-vinyl pyrrolidinone, and polyethylene glycol. Microdialysis sampling was used to test the effect of the hydrogel coating on glucose recovery. VEGF-releasing hydrogel-coated fibers increased vascularity and inflammation in the surrounding tissue after 2 weeks of implantation compared to hydrogel-coated fibers. DX-releasing hydrogel-coated fibers reduced inflammation compared to hydrogel-coated fibers and had reduced capsule vascularity compared to VEGF-releasing hydrogel-coated fibers. Hydrogels that released both VEGF and DX simultaneously also showed reduced inflammation at 2 weeks implantation; however, no enhanced vessel formation was observed indicating that the DX diminished the VEGF effect. At 6 weeks, there were no detectable differences between drug-releasing hydrogel-coated fibers and control fibers. From this study, hydrogel drug release affected initial events of the foreign body response with DX inhibiting VEGF, but once the drug depot was exhausted these effects disappeared.

Measurement of glucose and metabolites in subcutaneous adipose tissue during hyperglycemia with microdialysis at various perfusion flow rates.

BACKGROUND: Microdialysis-based glucose sensors have recently been introduced for monitoring glucose levels in diabetic patients. The flow rate by which the fluid sample is pumped through the microdialysis catheter varies in different studies. AIM: To study the effects of various flow rates on glucose and its metabolites sampled by microdialysis during an oral glucose tolerance test. MATERIAL, METHODS: Glucose, lactate, pyruvate and glycerol were measured with microdialysis in interstitial fluid of subcutaneous adipose tissue in twelve healthy young subjects before and during an oral glucose tolerance test using four different flow rates (0.3, 1, 2 and 5 microL/min) and a 30 mm dialysis membrane. RESULTS: At the basal fasting state the dialysate glucose obtained by 0.3 microL/min was equal to capillary glucose concentration. A decrease in dialysate glucose levels during the basal state was observed for higher flow rates but not for 0.3 microL/min, which indicates a depleting effect. The relative increase after OGTT was similar for capillary glucose and flow rate 0.3 microL/min but not for higher flow rates. CONCLUSION: The low microdialysis flow rate (0.3 microL/min) facilitates the capture of true interstitial glucose concentrations during glucose fluctuations. Thus this low flow rate is preferred in studies of local tissue metabolism.

Analyte flux through chronically implanted subcutaneous polyamide membranes differs in humans and rats.

The rat is commonly used to evaluate physiological responses of subcutaneous tissue to implanted devices. In vivo longevity of various devices and the biocompatibility of biomaterials depend on how adjacent tissue interacts. How closely the rat model predicts the human response has not been well characterized. The objective of this study was to compare rat and human subcutaneous foreign body responses by monitoring the biochemical environment at a polymer-tissue interface over 8 days using microdialysis. Polyamide microdialysis probes were implanted subcutaneously in humans and rats (n = 12). Daily microdialysis samples were analyzed for glucose, lactate, pyruvate, glycerol, and urea. Blood glucose was also monitored. Analyte concentrations differed significantly between rats and humans at the implant-tissue interface. There were also qualitative differences in the 8-day trends. For example, over 8 days, microdialysate glucose increased two- to fourfold in humans but decreased in rats (P < 0.001). This study reveals profound physiological differences at material-tissue interfaces in rats and humans and highlights the need for caution when extrapolating subcutaneous rat biocompatibility data to humans.

Urea as a recovery marker for quantitative assessment of tumor interstitial solutes with microdialysis.

Microdialysis is a technique that enables measurement of extracellular concentrations of unbound analytes. A small probe with a semipermeable membrane is implanted in tissue and constantly perfused. Small analytes in the interstitial fluid diffuse into the perfusate and are collected. Often, microdialysate concentrations of an analyte are only a fraction of the unbound concentrations in the extracellular space attributable to incomplete equilibration between these two compartments. Thus, it is necessary to determine the degree of equilibration between microdialysate and interstitium for each probe to accurately estimate concentrations. In this study, we investigated tissue urea as a solute to continually correct for nonequilibrium conditions. We used this method, along with relative diffusivities of urea and glucose, to monitor glucose levels before and during hyperglycemia as an example of how this method can be applied. No-net-flux experiments were performed on 10 anesthetized female rats with mammary adenocarcinomas. Microdialysis probes 1 cm in length with a molecular weight cutoff of M(r) 100,000 were used. Urea was added to the perfusate in concentrations of 0.83, 2.5, 5.0, and 13.33 mM. Microdialysate samples were collected every 15 min. For each rat, there was a linear relationship between the net urea concentration (outflow-inflow) and the urea concentration in the perfusate (inflow). Net flux should equal zero when perfusate and interstitial concentrations are equal. In an additional series of 13 rats, microdialysate samples were obtained before, during, and after administration of glucose at a dose of 1 g/kg. The interstitial tumor urea concentration was 7.8 +/- 0.3 mM compared with 6.2+/- 0.3 mM in plasma. There was a significant linear relationship between plasma urea (measured directly) and tumor urea (microdialysis measurement). Plasma urea concentrations were constant over time in all of the experiments, including those where hyperglycemia was induced. Hyperglycemia caused 7.7- and 3.6-fold increases in tumor and plasma glucose, respectively. There was no effect of hyperglycemia on tumor blood flow. Urea appears to be a useful low molecular weight relative recovery marker for tumor microdialysis. In combination with the determination of relative diffusivity between urea and the solute of interest, this calibration method may allow for quantitative measurements of tumor metabolites and unbound drugs.

Decreased analyte transport through implanted membranes: differentiation of biofouling from tissue effects.

Membrane biofouling and tissue changes in the foreign body response are known to cause detrimental reductions of analyte transport into implanted biosensors. The relative contribution of each phenomenon is unknown. Hollow fiber microdialysis probes were employed to assess the effect of subcutaneous implantation on glucose flux through polymeric membranes in rats over 8 days and to differentiate the transport effects of biofouling versus tissue changes. Three commercially available membranes were examined: poly(ether sulfone) (PES), polyacrylonitrile (PAN), and polycarbonate (PC). As measured by glucose recovery (the ratio of microdialysis glucose to blood glucose concentrations), transport through PES membranes was significantly less on day 2 than day 0 (39% decrease, p < 0.05) whereas PAN and PC showed no significant decreases in flux until day 8 (42 and 43%, respectively). Application of a transport model to glucose recovery data obtained before implantation in vivo and after explantation indicated that mass transport resistances originating from biofouling and tissue compartments increased between days 0 and 8. However, on average the biofouling layer adherent to the probe created substantially less resistance to glucose transport (12-24% of total) than did the tissue that surrounded the probe. These results suggested that future material developments for biosensors should be directed at understanding and modifying transport properties of tissues at the implant site.

Water-soluble treatments to enhance glucose permeability of protein-resistant polymer overlayers.

This study employed two water-soluble and nontoxic molecules, sucrose and glycerol, to enhance the permeability of PEG-PHEMA polymer gels coated onto 100 kDa molecular weight cutoff polyethersulfone (PES) microdialysis probes. Sucrose precoating of the probes prior to prepolymer coating prevented penetration of the prepolymer into the microdialysis membrane. Glycerol mixed with the prepolymer introduced porosity in the polymer coating upon curing. The sucrose and glycerol were completely removed by soaking in PBS after curing of the polymer coat on the probe tip. Polymer coated probe glucose permeability was tested by measuring glucose recovery from PBS solutions. Biocompatibility was assessed by measuring glucose recovery of polymer coated probes from heparanized whole porcine blood. Results show that the sucrose and glycerol treatments yielded polymer coated probes with glucose permeability nearly equal to bare probes when tested in PBS solution, but that this increased permeability was not observed when tested in whole blood. This suggests that the thickness of the polymer films (10-100 microm), while not a limiting factor in PBS solution, may have presented a diffusion barrier to glucose recovered from blood. Surprisingly, however, the polymer coated probes exhibited less thrombus formation that did the bare probes after blood exposure.

Methods for reducing biosensor membrane biofouling.

The deleterious effect that biofouling has on sensor stability is a serious impediment to the development of long term implanted biosensors. This paper reviews the surface modification strategies currently employed to minimize membrane biofouling of in vivo sensors. Nine sensor modifications are discussed herein: hydrogels, phospholipid-based biomimicry, flow-based systems, Nafion, surfactants, naturally derived materials, covalent attachments, diamond-like carbons, and topology.

Characterization of implantable biosensor membrane biofouling.

The material-tissue interaction that results from sensor implantation is one of the major obstacles in developing viable, long-term implantable biosensors. Strategies useful for the characterization and modification of sensor biocompatibility are widely scattered in the literature, and there are many peripheral studies from which useful information can be gleaned. The current paper reviews strategies suitable for addressing biofouling, one aspect of biosensor biocompatibility. Specifically, this paper addresses the effect of membrane biofouling on sensor sensitivity from the standpoint of glucose transport limitations. Part I discusses the in vivo and in vitro methods used to characterize biofouling and the effects of biofouling on sensor performance, while Part II presents techniques intended to improve biosensor biocompatibility.

MacAndrew Scale and sociodemographic correlates of adolescent alcohol and drug use.

To assess the utility of the MacAndrew Scale (MAC) of the MMPI as a screening device for problem drinking and drug use with adolescents, 403 ninth- through twelfth-graders were administered the MAC, a sociodemographic questionnaire, an alcohol/drug frequency questionnaire, the Adolescent Alcohol Involvement Scale, and the Adolescent Drug Involvement Scale. Stepwise multiple regression analyses showed the MAC to be the best single predictor (compared to sociodemographic variables) of alcohol/drug use for both males and females. MAC cutoff scores for differentiation between alcohol/drug abusers and nonabusers were obtained that were more efficient than base rate prediction. The results are discussed as supportive of the MAC's concurrent validity and of Jessor and Jessor's "problem behavior theory." Finally, implications for practice (e.g., early intervention and prevention) and future research are considered.