Bioactivation of Encapsulation Membranes Reduces Fibrosis and Enhances Cell Survival.

Encapsulation devices are an emerging barrier technology designed to prevent the immunorejection of replacement cells in regenerative therapies for intractable diseases. However, traditional polymers used in current devices are poor substrates for cell attachment and induce fibrosis upon implantation, impacting long-term therapeutic cell viability. Bioactivation of polymer surfaces improves local host responses to materials, and here we make the first step toward demonstrating the utility of this approach to improve cell survival within encapsulation implants. Using therapeutic islet cells as an exemplar cell therapy, we show that internal surface coatings improve islet cell attachment and viability, while distinct external coatings modulate local foreign body responses. Using plasma surface functionalization (plasma immersion ion implantation (PIII)), we employ hollow fiber semiporous poly(ether sulfone) (PES) encapsulation membranes and coat the internal surfaces with the extracellular matrix protein fibronectin (FN) to enhance islet cell attachment. Separately, the external fiber surface is coated with the anti-inflammatory cytokine interleukin-4 (IL-4) to polarize local macrophages to an M2 (anti-inflammatory) phenotype, muting the fibrotic response. To demonstrate the power of our approach, bioluminescent murine islet cells were loaded into dual FN/IL-4-coated fibers and evaluated in a mouse back model for 14 days. Dual FN/IL-4 fibers showed striking reductions in immune cell accumulation and elevated levels of the M2 macrophage phenotype, consistent with the suppression of fibrotic encapsulation and enhanced angiogenesis. These changes led to markedly enhanced islet cell survival and importantly to functional integration of the implant with the host vasculature. Dual FN/IL-4 surface coatings drive multifaceted improvements in islet cell survival and function, with significant implications for improving clinical translation of therapeutic cell-containing macroencapsulation implants.

Prolonged bioluminescence imaging in living cells and mice using novel pro-substrates for Renilla luciferase.

The prodrug or caged-luciferin strategy affords an excellent platform for persistent bioluminescence imaging. In the current work, we designed and synthesized ten novel pro-substrates for Renilla luciferase by introducing ester protecting groups of different sizes into the carbonyl group of the free luciferin 1. Taking advantage of intracellular esterases, lipases, and nucleophilic substances, the ester protecting groups were hydrolyzed, resulting in the release of a free luciferin and a bioluminescence signal turn-on. Among the tested pro-substrates, the butyryloxymethyl luciferin 7 exhibited low cytotoxicity and a prolonged luminescence signal both in cellulo and in vivo. Therefore, the butyryloxymethyl luciferin 7 can act as a promising substrate for noninvasive extended imaging in diagnostic and therapeutic fields.

Bioluminescent imaging of drug efflux at the blood-brain barrier mediated by the transporter ABCG2.

ATP-binding cassette (ABC) transporters are a group of transmembrane proteins that maintain chemical homeostasis through efflux of compounds out of organelles and cells. Among other functions, ABC transporters play a key role in protecting the brain parenchyma by efflux of xenobiotics from capillary endothelial cells at the blood-brain barrier (BBB). They also prevent the entry of therapeutic drugs at the BBB, thereby limiting their efficacy. One of the key transporters playing this role is ABCG2. Although other ABC transporters can be studied through various imaging modalities, no specific probe exists for imaging ABCG2 function in vivo. Here we show that D-luciferin, the endogenous substrate of firefly luciferase, is a specific substrate for ABCG2. We hypothesized that ABCG2 function at the BBB could be evaluated by using bioluminescence imaging in transgenic mice expressing firefly luciferase in the brain. Bioluminescence signal in the brain of mice increased with coadministration of the ABCG2 inhibitors Ko143, gefitinib, and nilotinib, but not an ABCB1 inhibitor. This method for imaging ABCG2 function at the BBB will facilitate understanding of the function and pharmacokinetic inhibition of this transporter.

Bioluminescence: imaging modality for in vitro and in vivo gene expression.

Molecular imaging offers many unique opportunities to study biological processes in intact organisms. Bioluminescence is the emission of light from biochemical reactions that occur within a living organism. Luciferase has been used as a reporter gene in transgenic mice but, until bioluminescence imaging was described, the detection of luciferase activity required either sectioning of the animal or excision of tissue and homogenization to measure enzyme activities in a conventional luminometer. Bioluminescence imaging (BLI) is based on the idea that biological light sources can be incorporated into cells and animal models artificially that does not naturally express the luminescent genes. This imaging modality has proven to be a very powerful methodology to detect luciferase reporter activity in intact animal models. This form of optical imaging is low cost and noninvasive and facilitates real-time analysis of disease processes at the molecular level in living organisms. Bioluminescence provides a noninvasive method to monitor gene expression in vivo and has enormous potential to elucidate the pathobiology of lung diseases in intact mouse models, including models of inflammation/injury, infection, and cancer.

Luciferin detection after intranasal vector delivery is improved by intranasal rather than intraperitoneal luciferin administration.

In vivo bioimaging of transgenic luciferase in the lung and nose is an expedient method by which to continually measure expression of this marker gene after gene transduction. Its substrate, luciferin, is typically injected into the peritoneal cavity before bioimaging. Here we demonstrate that, compared with intraperitoneal injection, intranasal instillation of luciferin confers approximately an order of magnitude increase in luciferase bioluminescence detection in both lung and nose. This effect was observed after administration of viral vectors based on adenovirus type 5, adeno-associated virus type 8, and gp64-pseudotyped HIV lentivirus and, to a lesser extent, after nonviral polyethylenimine (PEI)-DNA delivery. Detection increased relative to the concentration of luciferin; however, a standard concentration of 15 mg/ml was well beyond the saturation point. Compared with intraperitoneal injection, intranasal instillation yields about a 10-fold increase in sensitivity with an approximate 30-fold reduction in luciferin usage when bioimaging in the nasal and pulmonary airways of mice.

Characterization and real-time imaging of gene expression of adenovirus embedded silk-elastinlike protein polymer hydrogels.

Transient expression levels, vector dissemination and toxicities associated with adenoviral vectors have prompted the usage of matrices for localized and controlled gene delivery. Two recombinant silk-elastinlike protein polymer analogues, SELP-47K and SELP-415K, consisting of different lengths and ratios of silk and elastin units, were previously shown to be injectable hydrogels capable of matrix-mediated controlled adenoviral gene delivery. Reported here is a study of spatiotemporal control over adenoviral gene expression with these SELP analogues in a human tumor xenograft model of head and neck cancer using whole animal imaging. Real-time images of viral expression levels indicate that polymer concentration and polymer structure are predominant factors that affect viral release and, thus, viral transfection. Decrease in polymer concentration and increase in polymer elastin content results in greater release, probably due to changes in the network structure of the hydrogel. To better understand this relationship, macro- and microstructural properties of the hydrogels were analyzed using dynamic mechanical analysis (DMA) and transmission electron microscopy (TEM). The results confirm that the concentration and the elastin content of the protein polymer affect the pore size of the hydrogel by changing the physical constraints of the SELP fibril network and the degree of hydration of the SELP fibrils. The potential to modulate viral release using SELP hydrogel delivery vehicles that can be injected intratumorally by minimally invasive techniques holds significant promise for the delivery of therapeutic viruses.

In vivo bioluminescence imaging to evaluate estrogenic activities of endocrine disrupters.

Reporter gene technology is widely used to measure activity of hormone analogs, and bioluminescent in vitro assays have allowed rapid screening of numerous chemicals either to identify new agonists or antagonists of hormones or to detect the presence of endocrine disrupters in the environment. Stable bioluminescent cell lines have been established and they provide reproducible dose-response curves and accurate determination of in vitro efficiencies of various chemicals. In vivo, however, these molecules can be metabolized, bound by proteins, or stored in fats and thus could display efficiencies different from those observed in vitro. In vivo assays, such as the uterotrophic bioassay, require numerous sacrificed animals, and responses not only are dependent on an estrogenic action but also imply other factors. For a faster assay and to avoid the use of numerous animals, we developed an in vivo biosensor constituted of stable bioluminescent cells implanted in nude mice. MCF-7 bioluminescent cell lines were chosen since their proliferation is low in the absence of estrogen and the xenograft size can thus be stable for several weeks. Luciferase gene expression was monitored noninvasively with a cooled charge-coupled device camera. Quantitative analysis allowed us to compare in vitro and in vivo actions of different estrogenic compounds (estradiol, estrone) and endocrine disruptors (ethynylestradiol, genistein, octylphenol, and 2,4'-dichlorodiphenyldichloroethylene) in the same cell lines and to follow hormone action on a living animal as a function of time. Different administration protocols have been used and good correlation was observed for most products. However, we found that ethynylestradiol was the most efficient chemical when orally administered.

Firefly luciferin-activated rose bengal: in vitro photodynamic therapy by intracellular chemiluminescence in transgenic NIH 3T3 cells.

Photodynamic therapy (PDT) of cancer (1, 2) is a well-established treatment modality that uses light excitation of a photosensitive substance to produce oxygen-related cytotoxic intermediates, such as singlet oxygen or free radicals (3, 4). Although PDT is advantageous over other forms of cancer treatments because of its limited side effects, its main disadvantage is the poor accessibility of light to more deeply lying malignancies. External light sources such as lasers or lamps can be applied either noninvasively to reach tumors that lie well within the penetration depth of the light or in a minimally invasive fashion (interstitial treatments) in which optical fibers are placed intratumorally through needles. Even with the second approach, light distribution over the tumor is not homogeneous and nonidentified metastatic disease is left untreated. CL, the chemical production of light, is exemplified by firefly light emission mediated by the enzymatic (luciferase + ATP) oxidation of D-luciferin to oxyluciferin (5). This mobile light source is a targetable alternative to external sources of illumination. Here we show the in vitro photodynamic effect of rose bengal activated by intracellular generation of light, in luciferase-transfected NIH 3T3 murine fibroblasts.

Non-synaptic release of ATP by electrical stimulation in slices of rat hippocampus, cerebellum and habenula.

ATP is thought to be a fast neurotransmitter in the medial habenula region of the brain, and may be coreleased with other transmitters, for example with glutamate in the hippocampus. We monitored ATP release in rat brain slices using the bioluminescent indicator system luciferin-luciferase. Electrical stimulation of the hippocampus, cerebellum or habenula led to ATP release, but this release was calcium-independent and was not blocked by tetrodotoxin, or by other agents found to block ATP release from red blood cells. Although calcium-dependent ATP release may occur in response to electrical stimulation, it appears to be overwhelmed by calcium-independent release, which may result from electroporation of cells close to the stimulating electrode. Consistent with this, uptake into cells of the fluorescent dye Lucifer yellow was promoted by electrical stimulation. Our data undermine a previous suggestion, based on use of the luciferin-luciferase technique, that ATP is synaptically released with glutamate in the hippocampus.

Regulatory effects of ATP and luciferin on firefly luciferase activity.

ATP and luciferin are not only substrates of firefly luciferase, but can, in addition, modulate its activity. High concentrations of luciferin induce a conformational change of the enzyme that temporarily reduces the catalytic rate. Re-activation takes approx. 20 min and is independent of variation in the concentration of enzyme or ATP, but lengthens with increasing luciferin concentration. High concentrations of albumin reduce this luciferin effect. The kinetic properties of firefly luciferase determined from initial rates and at steady state after 1 min of catalysis have been analysed according to Michaelis-Menten kinetics. There is only one active site for each of the substrates. At steady state the Km and Vmax. values for both substrates are reduced in an uncompetitive manner. Hyperbolic Lineweaver-Burk plots indicate an activation by ATP probably by binding to an allosteric site. A model is presented which incorporates luciferin induced de- and re-activation effects. Experimental conditions to avoid the regulatory effects of substrates during ATP monitoring are proposed.

Continuous measurements of cytoplasmic ATP in single cardiomyocytes during simulation of the "oxygen paradox".

OBJECTIVE: It is now possible to monitor cytoplasmic ATP in single cardiomyocytes and it has recently been shown that cardiomyocytes exposed for several minutes to metabolic inhibitors undergo an abrupt rigor mediated shortening which coincides with a sudden fall in cytoplasmic ATP, from approximately 150 mumol.litre-1 to a few micromolar or less. The objective of this work was to monitor cytoplasmic ATP during simulated reoxygenation of a poisoned cardiomyocyte. METHODS: Firefly luciferase was injected into a single cell and the light signal generated when luciferin was superfused was monitored. Calibration of the signal is complicated by a transient enhancement of the signal (possibly the result of complex luciferase kinetics), and by uncertainties about cytoplasmic pH. RESULTS: The data indicate that millimolar levels of cytoplasmic ATP are restored within 1-2 min of cyanide removal. CONCLUSIONS: Cytoplasmic free calcium is known to rise after poisoned cells undergo shortening, so it is conceivable that the restoration of cytoplasmic ATP in a cell in which free calcium is at micromolar levels may provide a plausible cellular mechanism for the "oxygen paradox". Reoxygenation induces large amplitude, but slow, oscillations in free calcium which, together with the millimolar levels of ATP indicated here, could provide the stimuli for generating the uncoordinated mechanical forces that are prevalent in the oxygen paradox.

Inhibition by luciferin-luciferase reagents of aggregatory responses to excitatory agonists in washed platelet suspensions.

Addition of two commercial luciferin-luciferase reagents caused marked inhibition of the aggregatory response of washed human platelets to thrombin, ADP, vasopressin and platelet-activating factor (PAF). Analysis of the effects of the individual components of one of these reagents revealed that Mg2+, and to a lesser extent bovine serum albumin, was responsible for the observed inhibition. A modified luciferin/luciferase reagent has been designed on the basis of these data for use in washed platelet suspensions which causes minimal inhibition of the aggregatory and secretory responses to thrombin but which gives a near maximal luminescence yield.

Tetrodotoxin-resistant release of ATP from superfused rabbit detrusor muscle during electrical field stimulation in the presence of luciferin-luciferase.

The present study was carried out to assess the possible role of ATP in the noncholinergic, nonadrenergic transmission in the rabbit urinary bladder. When rabbit detrusor muscle strips were superfused with medium containing firefly luciferin-luciferase and stimulated transmurally at low stimulation parameters, tetrodotoxin-sensitive contractions were obtained but no release of ATP could be detected. However, at somewhat higher stimulation parameters, release of ATP was observed. This release of ATP was not diminished by tetrodotoxin indicating that ATP was not likely released as a result of propagated action potentials in nerves. Because contractions persisted in the presence of tetrodotoxin, it is possible that the ATP might have been released as a result of direct electrical stimulation of the muscle. These results do not support the idea that ATP is released as a neurotransmitter in the rabbit bladder.

An objective sperm cytotoxicity assay for male-specific antisera based on ATP levels of unlysed cells: application to assay of H-Y antigen.

We correlated the decrease in levels of ATP in spermatozoa with the extent of cytotoxicity elicited by antibodies against antigenic components on sperm. In the presence of concentrations of complement which did not cause cytolysis or influence the ATP content of epididymal sperm, addition of heat-in-activated sera from non-immunized mice, rats or rabbits did not result in sperm cytolysis or a fall in ATP content. In contrast, addition of rabbit anti-rat spermatocyte sera, which has previously been shown to react with rat spermatozoa (Tung, P.S. and Fritz, I.B. (1978) Dev. Biol. 64, 297-315), did cause sperm cytolysis and a decrease in ATP content. The titre of this antiserum for 50% cytolysis was between 1 : 128 and 1 : 256, as determined by the fall in ATP content. Using these criteria, we examined the cytotoxicity against sperm of different samples of anti H-Y sera. We examined the influence of monoclonal antibody against H-Y, mouse H-Y antisera and rat H-Y antisera raised in inbred females immunized with spleen cells from males of the same strains. In all cases, anti-H-Y lowered ATP levels and lysed sperm with a cytotoxic titre between 1 : 8 and 1 : 16. Measurements of the decrease in ATP content in sperm have been shown to provide an objective and reliable estimate of the percentage of spermatozoa lysed by H-Y antisera. Cytotoxic activity of H-Y antisera was removed by absorption with spleen cells from male mice but not by absorption with spleen cells from female mice.

Structure and evolution of calcium-modulated proteins.

This review suggests that the intracellular functions of calcium are best understood in terms of calcium's functioning as a second messenger. Further, when functioning as a second messenger, calcium completes its mission not by transferring charge nor by binding to lipid but by binding to specific targets, calcium-modulated proteins. This concept is broadly interpreted to include proteins involved in calcium transport. There is strong evidence that many, if not all, of these calcium-modulated proteins are homologs. Their structures and properties are contrasted to those of extracellular calcium-binding proteins which are not homologous to one another or to the intracellular calcium-modulated proteins. Finally, this line of thought leads to a suggestion of the evolutionary reason for the choice of calcium as the sole inorganic second messenger.