Scott R. Burks

Structure-dependent functional properties of Human Defensin 5

The mucosal epithelium secretes a variety of antimicrobial peptides that act as part of the innate immune system to protect against invading microbes. Here, we describe the functional properties of human defensin (HD) 5, the major antimicrobial peptide produced by Paneth cells in the ileum, in relation to its structure. The antimicrobial activity of HD‐5 against Escherichia coli proved to be independent of its structure, whereas the unstructured peptide showed greatly reduced antimicrobial activity against Staphylococcus aureus. We find that HD‐5 binds to the cell membrane of intestinal epithelial cells and induced secretion of the chemokine interleukin (IL)‐8 in a concentration‐ and structure‐dependent fashion. Incubation of HD‐5 in the presence of tumor necrosis factor alpha further increased IL‐8 secretion synergistically, suggesting that HD‐5 may act as a regulator of the intestinal inflammatory response.

Identification of Prostaglandin E2 Receptors Mediating Perinatal Masculinization of Adult Sex Behavior and Neuroanatomical Correlates

Prostaglandin E2 (PGE2) mediates the organization of male rat sexual behavior and medial preoptic area (MPOA) neuroanatomy during a sensitive perinatal window. PGE2 is up‐regulated in response to estradiol, and initiates a two‐fold increase in dendritic spines densities on neurons. All the four receptors for PGE2 and EP1‐4 are present in developing POA, a critical region controlling male sexual behavior. Previous studies explored that EP receptors are involved in PGE2‐induction of neonatal levels of spinophilin protein, a surrogate marker for dendritic spine formation, but did not assess behavioral masculinization. Here, we used two approaches, suppression of EP receptor expression with antisense oligonucleotides and activation of EP receptors with selective agonists, to test which receptors are necessary and sufficient, respectively, for the effects of PGE2 on behavior and neuronal morphology. In female rats, neonatal treatment with antisense oligonucleotides against EP2 or EP4 but not EP1 or EP3 completely prevented the expression of adult behavior organized by PGE2 exposure. The effects of ONO‐DI‐004, ONO‐AE‐259‐01, ONO‐AE‐248, and ONO‐AE1‐329 (EP1‐4 agonists respectively) were equivalent to PGE2 treatment, which suggests activating any EP receptor neonatally suffices in masculinizing sex behavior. When given alone, not all EP agonists increased neonatal POA spinophilin levels; yet giving each agonist neonatally increased adult levels. Moreover, adult spinophilin levels significantly correlated with two measures of male sexual behavior. The body of evidence suggests that EP2 and EP4 are both necessary and sufficient for PGE2‐induced masculinization of sex behavior, whereas EP1 and EP3 provide redundant roles.

2H,15N–Substituted Nitroxides as Sensitive Probes for Electron Paramagnetic Resonance Imaging

Electron paramagnetic resonance imaging (EPRI) using nitroxides is an emergent imaging method for studying in vivo physiology, including O(2) distribution in various tissues. Such imaging capabilities would allow O(2) mapping in tumors and in different brain regions following hypoxia or drug abuse. We have recently demonstrated that the anion of 3-carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl (2) can be entrapped in brain tissue to quantitate O(2) concentration in vivo. To increase the sensitivity of O(2) measurement by EPR imaging, we synthesized 3-carboxy-2,2,5,5-tetra((2)H(3))methyl-1-(3,4,4-(2)H(3),1-(15)N)pyrrolidinyloxyl (7). EPR spectroscopic measurements demonstrate that this fully isotopically substituted nitroxide markedly improves signal-to-noise ratio and, therefore, the sensitivity of EPR imaging. The new isotopically substituted nitroxide shows increased sensitivity to changes in O(2) concentration, which will enable more accurate O(2) measurement in tissues using EPRI.

Exploration of prostanoid receptor subtype regulating estradiol and prostaglandin E2 induction of spinophilin in developing preoptic area neurons

The prostaglandin E2 (PGE2) mediates estradiol-induced masculinization of sexual behavior in the rat during a perinatal sensitive period. PGE2 induces formation of dendritic spines on preoptic area (POA) neurons and this synaptic pattern change is associated with the ability to express male sexual behavior as an adult. Whether PGE2 is released from astrocytes or neurons in the developing POA is unknown. To further understanding of how PGE2 induces dendritic spine formation at the cellular level, we have explored the PGE2 receptor subtype mediating this response. There are four receptors for PGE2, EP1, EP2, EP3 and EP4, each having unique but interacting signal transduction profiles. Treatment of newborn female rats with the EP receptor agonists iloprost, butaprost and sulprostone indicated that stimulation of both the EP2 and EP3 receptors significantly increased spinophilin, a protein whose levels positively correlate to the presence of dendritic spines and masculinization of the POA. Use of antisense oligonucleotides against the mRNA for each receptor reveals that either EP2 or EP3 receptor knockdown reduces spinophilin in PGE2- or estradiol-treated females, whereas reducing EP1 or EP4 receptor levels by the same means has a smaller but also significant effect. A developmental profile of EP receptor expression indicates EP1 in particular is elevated for the first few days of life, corresponding to the critical period for masculinization, whereas mRNA levels for the other three receptors remain relatively constant.

Very-low-frequency electron paramagnetic resonance (EPR) imaging of nitroxide-loaded cells.

Recent advances in electron paramagnetic resonance (EPR) imaging have made it possible to image, in real time in vivo, cells that have been labeled with nitroxide spin probes. We previously reported that cells can be loaded to high (millimolar) intracellular concentrations with (2,2,5,5‐tetramethylpyrrolidin‐1‐oxyl‐3‐ylmethyl)amine‐N,N‐diacetic acid by incubation with the corresponding acetoxymethyl (AM) ester. Furthermore, the intracellular lifetime (t1/e) of this nitroxide is 114 min—sufficiently long to permit in vivo imaging studies. In the present study, at a gradient of ∼50 mT/m, we acquire and compare EPR images of a three‐tube phantom, filled with either a 200‐μM solution of the nitroxide, or a suspension of cells preincubated with the nitroxide AM ester. In both cases, 3‐mm resolution images can be acquired with excellent signal‐to‐noise ratios (SNRs). These findings indicate that cells well‐loaded with nitroxide are readily imageable by EPR imaging, and that in vivo tracking studies utilizing such cells should be feasible. Magn Reson Med 58:850–854, 2007.

Optimization of labile esters for esterase–assisted accumulation of nitroxides into cells: A model for in vivo EPR imaging

Nitroxide-based electron paramagnetic resonance (EPR) imaging agents are useful quantitative probes of O2 concentration in vivo in real time. Lipophilic, labile alkanoyloxymethyl esters of nitroxides can cross the blood-brain barrier, and after hydrolysis, the corresponding anionic nitroxide is intracellularly entrapped at levels sufficient to permit O2 measurements. The utility of nitroxides as EPR imaging agents depends critically on their ability to accumulate in the brain to high levels. In this study, we systematically investigated the relationship between the structure of the alkanoyl moiety and the ability of the corresponding labile ester to deliver nitroxide intracellularly. We demonstrate, in a cultured cell model, that for nitroxide labile esters with unbranched alkanoyl chains, increasing the chain length improves intracellular loading. Moreover, by studying an isomeric series of labile esters, we conclude that branching of the alkanoyl chain drastically reduces intracellular loading. These structural insights improve our general ability to use labile esters to deliver carboxylates intracellularly, and suggest a strategy for enhancing delivery of nitroxide imaging agents across the blood-brain barrier in a living animal.

The effect of structure on nitroxide EPR spectral linewidth.

Nitroxides with narrow linewidths are essential for low-frequency EPR spectroscopy and in vivo EPR imaging. In developing a framework for designing narrow-line nitroxides, we sought to understand the unexpectedly narrow line width of 4-oxo-2,2,6,6-tetramethyl-1-piperidinyloxyl (5). Computational modeling revealed that the carbonyl double bond in the 4-position allows conformational diversity that results in the observed narrowing of the EPR spectral line. In view of this finding, we synthesized two new nitroxides bearing an exocyclic double bond: 4-methoxycarbonylmethylidene-2,2,6,6-tetramethyl-1-piperidinyloxyl (7) and 4-acetoxymethoxycarbonylmethylidene-2,2,6,6-tetramethyl-1-piperidinyloxyl (9). These nitroxides, like nitroxide 5, exhibited narrow linewidths-consistent with the results of modeling. Nitroxide 8 (4-carboxymethylidene-2,2,6,6-tetramethyl-1-piperidinyloxyl), as a prototype, allows for a variety of structural diversity, such as nitroxide 9,that can, for instance, target tissue compartments for in vivo EPR imaging.

In vivo EPR oximetry using an isotopically-substituted nitroxide: Potential for quantitative measurement of tissue oxygen.

Variations in brain oxygen (O2) concentration can have profound effects on brain physiology. Thus, the ability to quantitate local O2 concentrations noninvasively in vivo could significantly enhance understanding of several brain pathologies. However, quantitative O2 mapping in the brain has proven difficult. The electron paramagnetic resonance (EPR) spectra of nitroxides are sensitive to molecular O2 and can be used to estimate O2 concentrations in aqueous media. We recently synthesized labile-ester-containing nitroxides, such as 3-acetoxymethoxycarbonyl-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl (nitroxide 4), which accumulate in cerebral tissue after in situ hydrolysis, and thus enable spatial mapping of O2 concentrations in the mouse brain by EPR imaging. In an effort to improve O2 quantitation, we prepared 3-acetoxymethoxycarbonyl-2,2,5,5-tetra((2)H3)methyl-1-(3,4,4-(2)H3,1-(15)N)pyrrolidinyloxyl (nitroxide 2), which proved to be a more sensitive probe than its normo-isotopic version for quantifying O2 in aqueous solutions of various O2 concentrations. We now demonstrate that this isotopically substituted nitroxide is ∼2-fold more sensitive in vivo than the normo-isotopic nitroxide 4. Moreover, in vitro and in vivo EPR spectral-spatial imaging results with nitroxide 2 demonstrate significant improvement in resolution, reconstruction and spectral response to local O2 concentrations in cerebral tissue. Thus, isotopic-substituted nitroxides, such as 2, are excellent sensors for in vivo O2 quantitation in tissues, such as the brain.

Clearance and Biodistribution of Liposomally Encapsulated Nitroxides: A Model for Targeted Delivery of Electron Paramagnetic Resonance Imaging Probes to Tumors

Electron paramagnetic resonance (EPR) imaging using nitroxides as molecular probes is potentially a powerful tool for the detection and physiological characterization of micrometastatic lesions. Encapsulating nitroxides in anti-HER2 immunoliposomes at high concentrations to take advantage of the “self-quenching” phenomenon of nitroxides allows generation of robust EPR signals in HER2-overexpressing breast tumor cells with minimal background from indifferent tissues or circulating liposomes. We investigated the in vivo pharmacological properties of nitroxides encapsulated in sterically stabilized liposomes designed for long circulation times. We show that circulation times of nitroxides can be extended from hours to days; this increases the proportion of liposomes in circulation to enhance tumor targeting. Furthermore, nitroxides encapsulated in sterically stabilized anti-HER2 immunoliposomes can be delivered to HER2-overexpressing tumors at micromolar concentrations, which should be imageable by EPR. Lastly, after in vivo administration, liposomally encapsulated nitroxide signal also appears in the liver, spleen, and kidneys. Although these organs are spatially distinct and would not hinder tumor imaging in our model, understanding nitroxide signal retention in these organs is essential for further improvements in EPR imaging contrast between tumors and other tissues. These results lay the foundation to use liposomally delivered nitroxides and EPR imaging to visualize tumor cells in vivo.

Comparison of Two Nitroxide Labile Esters for Delivering Electron Paramagnetic Resonance Probes into Mouse Brain

In vivo quantitation of O(2) in brain has been hindered by a lack of suitable imaging modalities. Development of low-frequency electron paramagnetic resonance (EPR) spectrometers that can detect free radicals in animals in real time makes it feasible to image paramagnetic oximetry probes such as nitroxides in brain tissue. We have shown that masking the carboxyl group of 3-carboxy-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl (nitroxide 1) as an esterase-labile acetoxymethyl ester yields 3-acetoxymethoxycarbonyl-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl (nitroxide 2). Nitroxide 2 can cross the blood-brain barrier and is then hydrolyzed in situ by esterases to regenerate nitroxide 1, which becomes entrapped in brain tissue. Seeking to improve the loading of nitroxides into brain, we synthesized the more lipophilic pentanoyloxymethyl ester, 3-pentanoyloxymethoxycarbonyl-2,2,5,5-tetramethyl-1-pyrrolidinyloxyl (nitroxide 3). We report that the higher lipophilicity of nitroxide 3 does not significantly increase its ability to generate EPR signals in the mouse brain. Therefore, irrespective of whether nitroxide 2 or 3 was injected, similar levels of nitroxide were entrapped in brain tissue. These findings suggest that nitroxides 2 and 3 perform comparably well as proimaging agents for measuring O(2) distribution in brain.