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BACKGROUND: The Special Programme for Research and Training in Tropical Diseases recently launched a Mycobacterium tuberculosis strain bank (TDR-TB Strain Bank). OBJECTIVE: To describe the TDR-TB Strain Bank, the characterisation of strains, bank management and the procedure for releasing materials. RESULTS: The TDR-TB Strain Bank consists of 229 clinical M. tuberculosis isolates (single-colony derived cultures) plus five mycobacterial reference strains for purposes of identification. These are available as freeze-dried, viable strains or as heat-inactivated bacterial suspensions, quality controlled for purity, viability and authenticity. Isolates originated from diverse geographical settings and were selected for their resistance profiles against first- and second-line drugs. Low and high levels of resistance were determined by the minimum inhibitory concentrations of isoniazid, rifampicin, ethambutol, streptomycin, ofloxacin, kanamycin, capreomycin, ethionamide and para-aminosalicylic acid. Sequencing for drug resistance mutations was performed on the relevant sections of the rpoB, katG, inhA, embB, rpsL, rrs, gyrA and gyrB genes. Typing using lineage-defining loci of mycobacterial interspersed repetitive unit-variable number tandem repeats indicated that the most important genetic lineages were represented. CONCLUSIONS: The TDR-TB Strain Bank is a high quality bioresource for basic science, supporting the development of new diagnostics and drug-resistant detection tools and providing reference materials for laboratory quality management programmes.

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BACKGROUND: The Special Programme for Research and Training in Tropical Diseases established a specimen bank in 1999 to support the development and evaluation of new tuberculosis (TB) diagnostic tools. OBJECTIVE: To provide a narrative of the bank's development and discuss lessons learned, the bank's limitations and potential future applications. RESULTS: Collection sites were selected in high- and low-prevalence settings. Patients with TB symptoms, consenting to participate and to undergo human immunodeficiency virus testing were enrolled and diagnosed. Serum, sputum, saliva and urine samples were collected and sent to the bank's repositories. The bank has stocked 41,437 samples from 2524 patients at 11 sites worldwide. Ninety-five requests for specimens have been reviewed and 67 sets have been approved. Approved applicants have received sets of 20 or 200 samples. The bank allowed an evaluation of 19 commercial lateral flow tests and showed that none of them had broad global utility for TB diagnosis. CONCLUSIONS: The establishment and development of the specimen bank have provided a wealth of experience. It is fulfilling a need to provide quality specimens, but the type and number of samples may not fulfil the demands of future end-users. Plans are underway to review the mechanisms of specimen collection and distribution to maximise their impact on product development.

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A major problem with implantable sensors is their short in vivo lifetime, due to strong tissue reactions (i.e., inflammation and fibrosis) caused by the implant and the failure of sensor components. The tissue reactions to the sensor, the biocompatibility of components, and the function of the sensor must be evaluated by using in vivo models. Current methods of in vivo biosensor testing are time- and labor- intensive and expensive. In addition, the results often vary on the basis of the surgical skills of the investigator. The in ova chorioallantoic membrane (CAM) of the developing chicken embryo was previously developed in our laboratory as a novel in vivo system to test biomaterials. In this new article, we describe a novel approach for testing biosensors in vivo using the ex ova CAM model as an alternative to the traditional mammalian models. Fertilized chicken eggs were incubated for 3 days in ova and then transferred into a petri dish (ex ova) for further incubation at 37 degrees C and 80% humidity. After 1 week of incubation, acetaminophen biosensors, used as model sensors, were placed on top of the CAM and allowed to incorporate for 1 week. Biosensors were then tested for their sensitivity to acetaminophen. CAM venules were injected with 0.2 mL of a 3.6 mM acetaminophen solution. The current produced by the sensor reflected the change in blood acetaminophen levels. Sensors were also assessed by using gross and histological evaluations. We previously reported on the similarity of the tissue response of the CAM with the mammalian models. The low cost, simplicity, and possibility to continuously visualize the sensor test site through a cell culture dish make this animal model particularly attractive for the rapid in vivo screening of biosensors.

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Current in vivo models for testing biomaterials are time and labor intensive as well as expensive. This article describes a new approach for testing biomaterials in vivo using the chorioallantoic membrane (CAM) of the developing chicken embryo, as an alternative to the traditional mammalian models. Fertilized chicken eggs were incubated for 4 days, at which time a small window was cut in the shell of the egg. After 1 week of incubation, the CAM received several test materials, including the endotoxin LPS, a cotton thread and a Silastic tubing. One day and 1 week later, the tissue response to the test materials was assessed using gross, histological, and scanning electron microscope evaluations. The inflammatory response of the chorioallantoic membrane to biomaterials was fully characterized and found to be similar to that of the mammalian response and was also seen to vary according to test materials. We also found that the structure and geometry of the test materials greatly influenced the incorporation of the samples in the CAM. The similarity of the tissue response of the CAM with the mammalian models, plus the low cost, simplicity, and possibility to continuously visualize the test site through the shell window make this animal model particularly attractive for the rapid in vivo screening of biomaterials.

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The purpose of this research effort was to evaluate in vivo a newly developed dexamethasone/PLGA microsphere system designed to suppress the inflammatory tissue response to an implanted device, in this case a biosensor. The microspheres were prepared using an oil/water (O/W) emulsion technique. The microsphere system was composed of drug-loaded microspheres (including newly formulated and predegraded microspheres) and free dexamethasone. The combination of the drug and drug-loaded microspheres provided burst release of dexamethasone followed by continuous release from days 2-14. Continuous release to at least 30 days was achieved by mixing predegraded and newly formulated microspheres. The ability of our mixed microsphere system to control tissue reactions to an implant then was tested in vivo using cotton thread sutures to induce inflammation subcutaneously in Sprague-Dawley rats. Two different in vivo studies were performed, the first to find the dosage level of dexamethasone that effectively would suppress the acute inflammatory reaction and the second to show how effective the dexamethasone delivered by PLGA microspheres was in suppressing chronic inflammatory response to an implant. The first in vivo study showed that 0.1 to 0.8 mg of dexamethasone at the site minimized the acute inflammatory reaction. The second in vivo study showed that our mixed microsphere system suppressed the inflammatory response to an implanted suture for at least 1 month. This study has proven the viability of microsphere delivery of an anti-inflammatory to control the inflammatory reaction at an implant site.

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The purpose of this research was to develop polylactic-co-glycolic acid (PLGA) microspheres for continuous delivery of dexamethasone for over a 1-month period, in an effort to suppress the acute and chronic inflammatory reactions to implants such as biosensors, which interfere with their functionality. The microspheres were prepared using an oil-in-water emulsion technique. The oil phase was composed of 9:1 dichloromethane to methanol with dissolved PLGA and dexamethasone. Some microspheres were predegraded for 1 or 2 weeks. Ten percent of polyethylene glycol was added to the oil phase in alternative formulations to delay drug release. The in vitro release studies were performed in a constant temperature (37 C) warm room, in phosphate-buffered saline at sink conditions. Drug loading and release rates were determined by HPLC-UV analysis. The standard microsphere systems did not provide the desired release profile since, following an initial burst release, a delay of 2 weeks occurred prior to continuous drug release. Predegraded microspheres started to release dexamethasone immediately but the rate of release decreased after only 2 weeks. A mixed standard and predegraded microsphere system was used to avoid this delay and to provide continuous release of dexamethasone for 1 month.

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Multilayered films of humic acids (HAs) (naturally occurring biopolymers) were investigated as a potential semipermeable membrane for implantable glucose sensors. These films were grown using a layer-by-layer self-assembly process of HAs and oppositely charged ferric ions. The growth of these assemblies exhibited strong dependence on the pH and ionic strength of HAs solutions, which correlated with the degree of ionization of the carboxyl groups and neutralization-induced surface spreading. Quartz crystal microbalance (QCM) and ellipsometric studies have shown repeatable, stepwise increase in mass (as high as 5.63 microg/cm(2)) and in film thickness (ca. 24.3 nm per layer) for these assemblies. The permeability of glucose through these membranes can be regulated by varying the number of self-assembled HAs/Fe(3+) layers. Moreover, a 200 nm thick HAs/Fe(3+) film (in its hydrated state) had a shear modulus of about 80 MPa, implying stability upon implantation. These films were determined to be biocompatible since in vivo studies indicated only mild tissue reaction along with some neovascularization.

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BACKGROUND: The degradation of the glucose oxidase (GOD) enzyme, commonly used in the construction of glucose sensors has been of concern for scientists for decades. Many researchers have found that GOD deactivates over time, mostly due to H2O2 oxidation. This decay can lead to the eventual failure of the sensor. However, these findings are controversial, because other researchers did not find this degradation. METHODS: The goal of this study was twofold. The first goal was to evaluate the in vitro and in vivo stability of two commercially available GOD enzymes and the second goal was to evaluate Nafion as a protective coating of GOD. Crosslinked GOD samples were sandwiched between two 10-microm pore polycarbonate membranes (Nafion coated or uncoated) and placed in custom designed Lexan chambers. Chambers were then exposed to a total of five different environments: Dulbecco's Modified Eagle Medium (DMEM) or phosphate buffered saline (PBS) with and without a 5.6-mM glucose concentration, as well as the subcutaneous in vivo environment of 12 rats. After a period of up to 4 weeks, chambers were retrieved, opened, and tested for enzyme activity using a three-electrode system. RESULTS: Enzyme activity showed only a slight decrease when exposed to DMEM and PBS without glucose. A more dramatic decrease in activity was observed in enzymes exposed to PBS and DMEM with 5.6 mM glucose. The in vivo environment also caused a significant decrease in enzyme activity, but the decrease was lower than for the in vitro environment with glucose conditions. CONCLUSION: The presence of glucose in vitro and in vivo led to the production of H2O2, suggesting this to be the main agent responsible for enzyme degradation. The use of a Nafion coating did not provide any additional protection.

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An electrostatic layer-by-layer deposition technique was employed for the formation of thin films consisting of alternating layers of perfluorinated ionomer (Nafion) and ferric ions. UV-vis spectroscopic and ellipsometric data indicate a stepwise growth that in certain cases is as high as 47 nm per dip cycle. The growth characteristics of these assemblies can be correlated with Nafion's hydrodynamic radius, iron content, as well as the ionic strength and pH of Nafion and the wash solution. When these assemblies were compared to cast Nafion films, they exhibit the following advantages: (i) increased hydrolytic stability, attained without thermal treatment required for pristine Nafion films, and (ii) resistance to calcification, by more than an order of magnitude. These results, along with the ability to control glucose permeability by varying the number of Nafion/Fe3+ layers, could prove vital in prolonging the lifetime of implantable biosensors.

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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.

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Since the perfluorosulfonated ionomer Nafion, commonly used for the protection of biosensors, experiences calcification in a biological environment, we evaluated the efficacy of preincubating Nafion membranes in a FeCl3 solution to reduce the number of nucleation sites responsible for the growth of the calcium phosphate crystals. Nafion membranes were prepared and divided into two groups. In the first group, the Nafion membranes were pre-incubated in 0.1 M FeCl3 for a 24 h period. In the second group, no pre-incubation took place. All membranes were placed in a culture medium for a period of up to 4 weeks. All membranes were then examined for changes in: (1) their surface topography (using scanning electron microscopy (SEM)); (2) their near surface chemical properties (using energy dispersive X-ray (EDX)); and (3) their permeability to glucose. The membranes that were not pre-incubated in FeCl3 showed significant cracking of the Nafion surface, extensive calcium phosphate deposits and a resulting decrease in permeability. In contrast, the membranes treated with FeCl3 showed almost no cracking, very little calcium phosphate deposits and no change in permeability to glucose. This study demonstrated that FeCl3 significantly reduces calcification of Nafion and thus should help in preserving the in vivo function of implantable biosensors that utilize Nafion in their design.

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The effects of the biological environment on the perfluorosulfonated ionomer Nafion membrane were investigated. Nafion membranes thermally annealed at 120 degrees C and kept in culture medium or implanted subcutaneously in rats showed extensive cracking after 4 weeks. In membranes annealed at 150 degrees C, cracking was reduced, but not eliminated. Deposits of calcium phosphates in the membrane were identified. These deposits appeared to be responsible for the cracking of the membranes, but the precise mechanism was unclear. The permeability to glucose of Nafion membranes annealed at 120 degrees C increased at 1 week and then decreased during the 3 following weeks. However, the cracking, protein adhesion, and mineralization of the membranes made the results difficult to interpret. This study revealed that mineralization of Nafion occurs in the biological environment, resulting in cracking and changes in permeability. Modifications to prevent the mineralization of Nafion are necessary to make it suitable for use in the implantable glucose sensor.

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This paper introduces a newly developed 2-channel bioartificial pancreas for the treatment of type I diabetes. In this device, insulin secreting cells (islets of Langerhans) are placed between 2 semi-permeable membranes that form a flat rectangular chamber. One side of the chamber is in contact with blood from an artery and the other side with blood from a vein. In addition to the diffusive transfer of glucose and insulin, the pressure difference between the artery and the vein creates an ultrafiltration flux through the islet chamber. This diffusion and ultrafiltration flux carries glucose from the artery to the islets, and insulin from the islets to the vein. The bioartificial pancreas has a compact geometry and the blood channels were designed to prevent blood clotting. The predicted insulin release in response to a square-wave and a triangular-wave glucose stimulation is given for the bioartificial pancreas of dimensions necessary for implantation in diabetic patients.

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In this paper we report that curing at 120 degrees C can be used to improve the in vivo durability of a miniaturized glucose sensor with an outer coating of the Dupont perfluorinated ionomer, Nafion. Sensors based on glucose oxidase trapped in an albumin/glutaraldehyde matrix were able to withstand curing at 120 degrees C without noticeable change in electrode sensitivity (+/- 22% SD). Curing above 120 degrees C caused a gradual decline in sensitivity, with no sensitivity seen at 170 degrees C. Curing Nafion at 120 degrees C eliminated ascorbic acid and urea interferences and improved selectivity for glucose against uric acid and acetaminophen, compared to room temperature-cured Nafion coatings. The Nafion film reduced O2 demand by the sensor, so the signal was O2 independent across a partial pressure range of 8-140 mmHg. Several of the fully assembled, heat-cured, needle-type glucose sensors remained functional for at least 10 days after subcutaneous implantation in dogs, without degradation of their sensitivity (average 3 nA/mM in vivo at 37 degrees C and 6 nA/mM in vitro at 37 degrees C).

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To assess the effect of the biological response to implanted Ag/AgCl reference electrodes on the electrode stability, uncoated and polymer-coated Ag/AgCl electrodes were implanted subcutaneously in rats. After 1 week of implantation, uncoated Ag/AgCl electrode potentials, measured in 0.1 M KCl, shifted by about -180 mV, and both voltammetry and electron microscopy showed that all the AgCl was removed. The electrodes could be significantly protected by coating with polyurethane or a perfluorinated ionomer (Nafion) cured at 120 degrees C for 1 h. Electron micrographs showed the 120 degrees C cured Nafion and polyurethane coatings remained intact over 2 weeks of implantation. Following 2 weeks of implantation the cured, Nafion-coated electrodes' potentials were shifted by -15 +/- 7 mV relative to the initial values. Voltammetry showed that they were still not polarizable. The current densities obtained with the coated reference electrodes are sufficient for their use as counter/pseudoreference electrodes with implantable two-electrode glucose sensor systems. The tissue response to coated electrodes was minimal in comparison to the response to uncoated reference electrodes.

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We have developed an implantable glucose sensor based on a new tri-layer membrane configuration. The needle-type sensor integrates a Pt working electrode and a Ag/AgCl reference electrode. Its size is equivalent to a 25 gauge needle (0.5 mm in diamater). Poly (o-phenylenediamine) was used as an inner coating to reduce interference by small compounds present in the body fluids, and the perfluorinated ionomer, Nafion as a biocompatible, protective, outer coating. Glucose oxidase trapped in an albumin/glutaraldehyde matrix was sandwiched between these coatings. In vitro tests in buffer showed the sensors had a good selectively, a sensitivity of about 25 nA/mM, and a 90% response time of 33 s. Stabilization of the current following polarization required 10 to 30 min in vitro and 30 to 40 in vivo. Although these sensors remained stable for many weeks in saline solution, their implantation in animals resulted in the degradation of the protective Nafion outer coating, which in turn, led to the failure of the incorporated reference electrode. We demonstrated that if unprotected, the AgCl layer of the reference electrode rapidly dissolves in the biological environment. However, we later showed that in vivo degradation of Nafion can be prevented by heat curing. When heat cured sensors were subcutaneously implanted in dogs, the sensors' signal closely followed the plasma glucose level during glucose tolerance tests. The response of the sensors implanted in dogs was retained for 10 days.

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A miniature, needle-type glucose sensor based on a new trilayer membrane configuration has been prepared and evaluated both in vitro and in vivo. The perfluorinated ionomer, Nafion, was used as a protective, biocompatible, outer coating, and poly(o-phenylenediamine) as an inner coating to reduce interference by small, electroactive compounds. Glucose oxidase immobilized in a bovine serum albumin matrix was sandwiched between these coatings. The entire sensor assembly of Pt working electrode and Ag/AgCl reference electrode was 0.5 mm in diameter and could be inserted subcutaneously through an 18-gauge needle. The sensor current closely followed the plasma glucose level during a glucose tolerance test in active dogs, with a delay of 3 min, corresponding to the known lag time for subcutaneous glucose levels. The sensor remained functional after 1 week of implantation, but failed after 2 weeks due to degradation of the reference electrode. In vitro tests in pH 7.4 buffer or whole blood show the sensors have good selectivity, sensitivity of about 25 nA/mM, precision of 2-5%, and a 90% response time of 33 s. Stabilization following polarization requires 10-30 min in vitro and 30-40 min in vivo.

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With the use of a technique based on the detachment of single cells by suctioning of the cells with a glass micropipette, the authors studied human endothelial cell adhesion on 4 different surfaces (Fluorocarbon FC 721, polyester, nylon, glass) versus time of contact in phosphate buffered solution and a complete culture medium. It was observed that the force of detachment from solid surfaces of endothelial cells increases significantly with time and with increasing substrate surface tension in PBS and decreasing substrate surface tension in complete culture medium. This dependence of cell-fiber interactions with surface tension of the fibers demonstrates that cell adhesion in the authors' experimental conditions in primarily controlled by surface tension.

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