Increased vaccine tolerability and protection via NF-κB modulation.

Improving adjuvant responses is a promising pathway to develop vaccines against some pathogens (e.g., HIV or dengue). One challenge in adjuvant development is modulating the inflammatory response, which can cause excess side effects, while maintaining immune activation and protection. No approved adjuvants yet have the capability to independently modulate inflammation and protection. Here, we demonstrate a method to limit inflammation while retaining and often increasing the protective responses. To accomplish this goal, we combined a partial selective nuclear factor kappa B (NF-kB) inhibitor with several current adjuvants. The resulting vaccines reduce systemic inflammation and boost protective responses. In an influenza challenge model, we demonstrate that this approach enhances protection. This method was tested across a broad range of adjuvants and antigens. We anticipate these studies will lead to an alternative approach to vaccine formulation design that may prove broadly applicable to a wide range of adjuvants and vaccines.

Fabrication of Bragg gratings in planar PMMA: impact of UV dosage and thermal annealing.

We report on the fabrication of planar Bragg gratings in polymer substrates and study the impact of the UV dosage and a subsequent thermal annealing on the reflectivity of the gratings and the full width at half maximum bandwidth of the reflected spectra. In addition, the influence of the grating length is investigated, showing that gratings as short as 4 mm continuously exhibit good reflection properties, facilitating miniaturized sensor designs. Moreover, we highlight that the polymer Bragg gratings exhibit a remarkable stable reflected spectrum for over two years. Finally, the experimentally determined spectral characteristics of the Bragg gratings are compared to simulated results revealing excellent agreement.

Compressive and tensile strain sensing using a polymer planar Bragg grating.

A polymer planar Bragg grating sensor is used for measuring both mechanical compressive and tensile strain. The planar waveguide with integrated Bragg grating is fabricated in bulk Polymethylmethacrylate in a single writing step using combined amplitude and phase mask technique. After butt coupling of a single-mode optical fiber the planar structure can be applied for measuring both mechanical tensile and compressive strain alongside the integrated waveguide without the need of further modifications. In this respect, we particularly report for the first time compressive strain measurements using a polymer Bragg grating. Furthermore, the sensitivity of the sensor against tensile and compressive strain, its reproducibility and hysteresis are investigated and discussed.

Allylated cyclodextrins as effective affinity materials in chemical sensing of volatile aromatic hydrocarbons using an optical planar Bragg grating sensor.

We report on the application of perallyl-substituted α-, ß- and γ-cyclodextrins to an optical planar Bragg grating refractive index sensor for the effective sensitization of the sensor for airborne volatile aromatic hydrocarbons. Thereby, the emphasis of this work lies on the comparison of the different cyclodextrin types regarding their suitability as affinity material assessed by the sensors sensitivity and response behavior. The opto-chemical sensor device showed an immediate and quick response to the application of the investigated analytes benzene, toluene and m-xylene as well as a linear dependence on the concentration of those analytes. Studies on the sensors sensitivity depending on the applied cyclodextrin types revealed a generally higher sensitivity for the sensor sensitized with perallyl-substituted ß-cyclodextrins. Here, the sensor systems detection limit was found to 60±4 ppm for benzene, 18±3 ppm for toluene and 3.8±0.5 ppm for m-xylene. The response time and recovery time were found to approximately 30s and 40s, respectively, depending on the applied cyclodextrin and the chosen analyte.

Polymer planar Bragg grating sensor for static strain measurements.

We report on a new optical strain sensor based on a polymer planar Bragg grating (PPBG). The sensor consists of commercially available bulk Polymethlymethacrylate with a UV-inscribed optical waveguide as well as a Bragg grating, both of which are fabricated simultaneously in a single writing step. Upon axial strain, the Bragg wavelength reveals a quasi-instantaneous spectral red shift that depends linearly on the mechanical load with a sensitivity of 2.95 pm/µÎµ. The relative reflected intensity of the PPBG remains constant in the investigated load region.

Planar Bragg grating in bulk polymethylmethacrylate.

We report on a one-step writing process of a planar waveguide including a Bragg grating structure in bulk Polymethylmethacrylate (PMMA). A KrF excimer laser and a phase mask covered by an amplitude mask were used to locally increase the refractive index in PMMA and thereby generate simultaneously the planar waveguide and the Bragg grating. Our results show a reflected wavelength of the Bragg grating of about 1558.5 nm in accordance to the phase mask period. The reflectivity of the grating is about 80%. Initial characteristics of the Bragg grating structure towards humidity are investigated.

Sedentary behavior: target for change, challenge to assess.

Sedentary behavior is not a new topic, but trying to examine the direct links between sedentary behavior and health outcomes, independent of time spent in moderate- and vigorous-intensity physical activity, is a relatively new addition to the relationships between physical activity and health. Defining sedentary behavior as a risk factor and target for intervention opens up novel avenues for disease prevention and health promotion. The relationship between sedentary behavior and obesity is complex and not well understood, but the increased risk of disease due to sedentary behavior may be even greater in obese patients. Objective measurement of sedentary behavior is an important link in being able to understand the real effects of being sedentary, and a few measurement devices are described. Interventions targeting sedentary behavior should reduce total sedentary time, break long bouts of sitting with intermittent activity and encourage light-intensity activity throughout the day. New technologies can both measure and deliver an intervention aimed at reducing sitting time, the most common category of sedentary behavior. An optimal activity profile will include minimal amounts of sedentary behavior, in addition to regular physical activity and healthy sleep patterns.

Predicting Adult Pulmonary Ventilation Volume and Wearing Compliance by On-Board Accelerometry During Personal Level Exposure Assessments.

BACKGROUND: Metabolic functions typically increase with human activity, but optimal methods to characterize activity levels for real-time predictions of ventilation volume (l/min) during exposure assessments have not been available. Could tiny, triaxial accelerometers be incorporated into personal level monitors to define periods of acceptable wearing compliance, and allow the exposures (µg/m3) to be extended to potential doses in µg/min/kg of body weight? OBJECTIVES: In a pilot effort, we tested: 1) whether appropriately-processed accelerometer data could be utilized to predict compliance and in linear regressions to predict ventilation volumes in real time as an on-board component of personal level exposure sensor systems, and 2) whether locating the exposure monitors on the chest in the breathing zone, provided comparable accelerometric data to other locations more typically utilized (waist, thigh, wrist, etc.). METHODS: Prototype exposure monitors from RTI International and Columbia University were worn on the chest by a pilot cohort of adults while conducting an array of scripted activities (all <10 METS), spanning common recumbent, sedentary, and ambulatory activity categories. Referee Wocket accelerometers that were placed at various body locations allowed comparison with the chest-located exposure sensor accelerometers. An Oxycon Mobile mask was used to measure oral-nasal ventilation volumes in-situ. For the subset of participants with complete data (n= 22), linear regressions were constructed (processed accelerometric variable versus ventilation rate) for each participant and exposure monitor type, and Pearson correlations computed to compare across scenarios. RESULTS: Triaxial accelerometer data were demonstrated to be adequately sensitive indicators for predicting exposure monitor wearing compliance. Strong linear correlations (R values from 0.77 to 0.99) were observed for all participants for both exposure sensor accelerometer variables against ventilation volume for recumbent, sedentary, and ambulatory activities with MET values ~<6. The RTI monitors mean R value of 0.91 was slightly higher than the Columbia monitors mean of 0.86 due to utilizing a 20 Hz data rate instead of a slower 1 Hz rate. A nominal mean regression slope was computed for the RTI system across participants and showed a modest RSD of +/-36.6%. Comparison of the correlation values of the exposure monitors with the Wocket accelerometers at various body locations showed statistically identical regressions for all sensors at alternate hip, ankle, upper arm, thigh, and pocket locations, but not for the Wocket accelerometer located at the dominant-side wrist location (R=0.57; p=0.016). CONCLUSIONS: Even with a modest number of adult volunteers, the consistency and linearity of regression slopes for all subjects were very good with excellent within-person Pearson correlations for the accelerometer versus ventilation volume data. Computing accelerometric standard deviations allowed good sensitivity for compliance assessments even for sedentary activities. These pilot findings supported the hypothesis that a common linear regression is likely to be usable for a wider range of adults to predict ventilation volumes from accelerometry data over a range of low to moderate energy level activities. The predicted volumes would then allow real-time estimates of potential dose, enabling more robust panel studies. The poorer correlation in predicting ventilation rate for an accelerometer located on the wrist suggested that this location should not be considered for predictions of ventilation volume.

Developmental regulation of EphA4 expression in the chick auditory brainstem.

The avian auditory brainstem nuclei nucleus magnocellularis (NM) and nucleus laminaris (NL) display highly precise patterns of neuronal connectivity. NM projects tonotopically to the dorsal dendrites of ipsilateral NL neurons and to the ventral dendrites of contralateral NL neurons. The precision of this binaural segregation is evident at the earliest developmental stage at which connections can be observed. We have begun to examine the possibility that Eph receptor tyrosine kinase signaling is involved in establishing these spatially segregated connections. The expression of the EphA4 tyrosine kinase was examined at several developmental stages. EphA4 is expressed in rhombomere 5, which contains progenitors for both NM and NL. In this rhombomere, the labeling becomes striped during the time that precursor cells migrate to the auditory anlage. At the precise time when NM-NL projections are forming, EphA4 expression in NL is asymmetric, with markedly higher expression in the dorsal NL neuropil than in the ventral neuropil, suggesting a possible role in guiding growing axons to the appropriate region. At later embryonic ages EphA4 expression is symmetric around NL, and is absent in NM. As auditory function matures, EphA4 expression decreases so that by 4 days after hatch no EphA4 antibody labeling is evident in the auditory brainstem nuclei.

19F-NMR studies of retinol transfer between cellular retinol binding proteins and phospholipid vesicles.

The cellular retinol binding proteins, CRBP and CRBP II, are implicated in the cellular uptake of retinol and intracellular trafficking of retinol between sites of metabolic processing. 19F-NMR studies of retinol transfer between CRBP and CRBP II and phospholipid vesicles, using either fluorine-labeled ligand or protein, demonstrated that there was significantly more transfer of retinol from CRBP II to lipid vesicles than from CRBP. Differences in how readily protein-bound retinol is released to lipid bilayers may lead to differences in how these two proteins modulate intracellular retinol metabolism.

Nuclear magnetic resonance studies demonstrate differences in the interaction of retinoic acid with two highly homologous cellular retinoic acid binding proteins.

Cellular retinoic acid binding protein-I (CRABP-I) and cellular retinoic acid binding protein-II (CRABP-II) are highly homologous, 15 kDa proteins which bind all-trans-retinoic acid. In the adult, CRABP-II is expressed predominately in the epidermis, while CRAPB-I is expressed in a variety of tissues. To obtain structural information which could aid the design of more selective ligands, isotope-directed NMR methods were employed to observe the CRABP-bound conformation of 13C-labeled retinoic acid and to identify its contact points with neighboring amino acids. Analysis of HMQC, HMQC-TOCSY, and 13C-TOCSY-REVINEPT on CRABP-bound (2,3,6,7,8,9,10,11,19-13C)- and (1,4,5,8,9,16, 17,18,19-13C)-all trans-retinoic acid allowed the unambiguous assignment of all labeled protons and their attached 13C resonances. The volumes of 16 olefinic proton-methyl NOE cross-peaks measured from 30-ms 13C-(omega 2)-filtered 1H NOESY experiments were used to determine the conformations about the 6-, 8-, and 10-single bonds of the retinoic acid polyene chain. These spectra show qualitatively distinct NOE patterns for the two CRABPs. Measured cross-peak volumes for CRABP-II bound retinoic acid were well predicted by a single, static conformational having a 6-s torsion angle of -60 degrees skewed from a cis conformation. In contrast, for CRABP-I no single, static conformation was able to match the pattern of cross-peaks, suggesting motion about the 6-s bond. The measured cross-peaks were best described by 8-s and 10-s torsion angles of 180 degrees +/- 30 degrees, a trans configuration, for both proteins. The pattern of intermolecular NOESY cross-peaks between 13C-labeled protons in the ring portion of retinoic acid and protein protons were different between CRABP-I and CRABP-II. These differences coincide well with nearby amino acid substitutions in the recently reported X-ray structures of crystalline CRABP-I and CRABP-II and may assist rational design of selective ligands.

Measurement of subnanomolar retinoic acid binding affinities for cellular retinoic acid binding proteins by fluorometric titration.

Cellular retinoic acid binding protein I (CRABP-I) and cellular retinoic acid binding protein II (CRABP-II) are small, cytoplasmic proteins which bind all-trans-retinoic acid with high affinity. Both of these proteins belong to a family of intracellular proteins which bind amphiphilic lipids, including fatty acids, bile salts, and retinoids. Because CRABP-I and -II exhibit different tissue distributions and differential transcriptional regulation, they are proposed to serve different functions. The binding properties of mouse CRABP-I and -II purified from Escherichia coli were examined to further understand their role in intracellular retinoic acid processing. Fluorescence titrations were performed using nanomolar protein concentrations, near the obtained dissociation constants, and analyzed by direct mathematical fitting to raw data, in order to extend the range and accuracy of binding constant determination. The apparent dissociation constants, K'd, of mouse CRABP-I and CRABP-II binding all-trans-retinoic acid were determined to be 0.4 +/- 0.3 nM and 2 +/- 1 nM respectively, stronger binding than previously reported. The K'd of mCRABP-I and mCRABP-II complexing with acitretin, a pharmacologically active synthetic retinoid used in the treatment of psoriasis, was 3 +/- 1 nM and 15 +/- 11 nM. Both CRABPs bound 9-cis-retinoic acid with a K'd of roughly 200 nM, and neither exhibited significant binding of 13-cis-retinoic acid.

NMR studies of fluororetinol analogs complexed to two homologous rat cellular retinol-binding proteins.

Comparative 19F-NMR studies of fluororetinol analogs with rat cellular retinol binding protein II (CRBP II) and rat cellular retinol-binding protein (CRBP) were performed to probe differences in the binding interactions of these two homologous proteins. Line shape analyses of 19F-NMR spectra of (E,E,Z,E)-6-fluoro-9-(4-methoxy-2,3,6-trimethylphenyl)-3,7-dimethyl- 2,4,6,8-nonatetren-1-ol (ligand 1), (E,E,Z,E)-6-fluoro-9-(2,2' dimethyl-6-methylcyclohexenyl)-3,7- dimethyl-2,4,6,8-nonatetren-1-ol (ligand 2), (E,Z,E,E)-5-fluoro-9-(2,2'- dimethyl-6-methylcyclohexenyl)-3,7-dimethyl-2,4,6,8-nonatetren+ ++-1-ol (ligand 3), when complexed with CRBP II at temperatures ranging from 0-45 degrees C, revealed that the 19F resonances corresponding to the bound ligand were in slow chemical exchange between two resonance frequencies. This was further supported by a 2D-NOESY exchange experiment. The kex at 25 degrees C was estimated from spectral simulation and fitting analyses to be 887 s-1, 1010 s-1 and 771 s-1 for CRBP II complexed 1, 2, and 3, respectively. In contrast, only a single absorption was observed for bound ligands complexed with rat CRBP over this temperature range, suggesting that the conformational dynamics of retinol binding are different for these two closely homologous proteins.

Characterization of the ligand binding domain of human retinoid X receptor alpha expressed in Escherichia coli.

In order to study the structural details of ligand protein interactions of the human retinoid X receptor alpha (hRXR alpha), the DEF and EF domains of the receptor were expressed as glutathione S-transferase (GST) fusion proteins in Escherichia coli. The fusion proteins were expressed at high levels and were affinity-purified by chromatography over glutathione-agarose. The DEF and EF domains were cleaved from the fusion proteins by digestion with thrombin. Retinoic acid binding was quantitated using two different methods. The apparent dissociation constant (Kd) and the stoichiometry of 9-cis-retinoic acid binding were performed by monitoring quenching of protein fluorescence. To directly compare the binding affinity of the E. coli-derived truncated hRXR alpha with full-length hRXR alpha expressed in transiently transfected COS cells, Scatchard analyses of [3H]9-cis-retinoic acid binding assays were performed. Both methods of analysis indicate that while the cleaved DEF peptide bound 9-cis-retinoic acid tightly, the cleaved EF peptide exhibited variable binding activity between preparations. By fluorimetric analysis, the Kd of the cleaved DEF peptide was estimated to be 3 +/- 0.5 nM with a stoichiometry of 1:1.1 +/- 0.1. By Scatchard analysis, the Kd values for [3H]9-cis-retinoic acid to the GST-hRXR alpha (DEF) peptide and the cleaved DEF peptide were estimated to be 1.8 nM and 5.6 nM, respectively. The estimated molecular mass from high speed sedimentation equilibrium experiments was 36 +/- 2 kDa for the apo-DEF peptide alone and 38 +/- 3 kDa for the holo-DEF peptide complexed with 9-cis-retinoic acid. This suggests that the recombinant ligand binding domain was predominantly in the monomer form. However, dimers of the cleaved DEF peptides were detected in chemical cross-linking experiments both in the presence and absence of 9-cis-retinoic acid. Since the purified E. coli-derived truncated hRXR alpha DEF peptide appears to fully retain its ligand binding activity, it should provide a useful model system for further structural analysis of ligand-protein interactions.

Aromatic retinoid Acid-derivatives are potent inducers of differentiation of neuroblastoma-cells - structure-function relationship and the involvement of the nuclear retinoic Acid receptors.

Twenty-two derivatives of retinoic acid (RA) were tested for their potency to induce in vitro morphological differentiation of SK-N-SH neuroblastoma cell line at concentration range of 0.1 nM to 10 muM. The results indicate that three derivatives Ro-13-4306; Ro-13-6307 and Ro-13-7410 were potent inducers of differentiation at 3-4 log lower concentration compared to all trans retinoic acid (ATRA). Other compounds such as Ro-08-8717 and Ro-40-6055, were totally inactive, and with the majority of compounds inducing differentiation at concentrations comparable to ATRA. Fourteen compounds were studied for their hydrophobicity properties and the empirical hydrophobicity index (EHI) was derived from the retention time on an HPLC reverse column. Most of the EIH values derived for the compounds tested fell within the expected differentiation potency (ID50) range. However, ATRA and 9-cis RA fell below the expected curve, i.e. were less active than predicted by their hydrophobicity properties, whereas Ro-13-6307 and Ro-13-4306 fell above the curve, and thus were more potent in inducing differentiation than was predicted from their hydrophobicity properties. Energy minimized molecular models for the theoretical crystal structures were also reconstructed by computer modelling for five compounds. The molecular models of the two most active compounds (13-4306 and 13-6307) had almost identical crystal structure which was slightly different from that of the less active compounds, ATRA and 13-7410. SK-N-SH cell line expressed mRNA transcripts for the nuclear RA receptor-alpha (RARalpha) and did not express the RARbeta or RARgamma receptor. Induction of differentiation with ATRA, or with Ro-13-7410 did not change the pattern of expression of mRNA transcripts of any of the nuclear receptors tested. However, 13-4306 and 13-6307 markedly reduced the expression of the 2.4 kb mRNA band of RAR-alpha. These results, taken together, suggest the increased potency of 13-6307 and 13-4306 might be the result of a different splicing patterns of the RAR-alpha mRNA transcripts.

Differential binding of retinol analogs to two homologous cellular retinol-binding proteins.

A comparative study of the interactions of rat cellular retinol-binding protein (CRBP) and cellular retinol-binding protein II (CRBP II) with a number of synthetic phenyl-substituted analogs of all-trans-retinol was performed using fluorescence and nuclear magnetic resonance analysis. These studies indicate that CRBP II is more sensitive to modifications of the ring moiety than CRBP. Removal of the two methyl substituents on the ring which are ortho to the polyene chain abolishes binding to CRBP II. Conformational analysis of the ligands indicates that these two methyl groups influence the planarity of the ligand. The identification of monospecific ligands may prove useful for studying the physiological roles of these two proteins.

Retinoic acid receptors and retinoid X receptors: interactions with endogenous retinoic acids.

The binding of endogenous retinoids and stereoisomers of retinoic acid (RA) to the retinoid nuclear receptors, RA receptor (RARs) and retinoid X receptors (RXRs), was characterized using nucleosol preparations from transiently transfected COS-1 cells. Among several stereoisomers of RA tested, including 7-cis-, 9-cis-, 11-cis-, 13-cis-, and all-trans-RA, only 9-cis-RA effectively competes with 9-cis-[3H]RA binding to the RXRs. Additionally, the endogenous retinoid trans-didehydro-RA (t-ddRA) does not interact with RXRs, whereas the 9-cis form of ddRA competes effectively. RXRs (alpha, beta, and gamma) bind 9-cis-RA with dissociation constants (Kd) of 15.7, 18.3, and 14.1 nM, respectively. In contrast to the selectivity of RXRs for 9-cis-RA, RARs bind both t-RA and 9-cis-RA with high affinity, exhibiting Kd values in the 0.2-0.7 nM range for both ligands. Unlike RARs, the cellular RA binding proteins CRABPI or CRABPII bind t-RA but do not bind 9-cis-RA. Consistent with the binding data, 9-cis-RA and 9-cis-ddRA transcriptionally activate both GAL4-RXR and GAL4-RAR chimeric receptors with EC50 values of 3-20 nM for 9-cis-RA and 9-cis-ddRA, whereas t-RA and t-ddRA efficiently activate only GAL4-RAR chimeric receptors. Thus, 9-cis forms of endogenous retinoids can contribute to the pleiotropic effects of retinoids by interacting with both the RARs and RXRs.

9-cis retinoic acid stereoisomer binds and activates the nuclear receptor RXR alpha.

Vitamin A (retinol) and its natural derivatives are required for many physiological processes. The activity of retinoids is thought to be mediated by interactions with two subfamilies of nuclear retinoic acid receptors, RAR and RXR. The RARs bind all-trans retinoic acid (t-RA) with high affinity and alter gene expression as a consequence of this direct ligand interaction. RXR alpha is activated by t-RA, yet has little binding affinity for this ligand. t-RA may be converted to a more proximate ligand that directly binds and activates RXR alpha, and we have developed a method of nuclear receptor-dependent ligand trapping to test this hypothesis. Here we report the identification of a stereoisomer of retinoic acid, 9-cis retinoic acid, which directly binds and activates RXR alpha. These results suggest a new role for isomerization in the physiology of natural retinoids.

Human biliary metabolites of isotretinoin: identification, quantification, synthesis, and biological activity.

1. The metabolites of isotretinoin (13-cis-retinoic acid, Accutane) were investigated in the bile of two patients with biliary T-tube drainage after administration of a single, oral, 80-mg dose of 14C-isotretinoin. Radioactivity measurements showed that the two patients excreted 22.7 and 17.1% of the dose in their bile in 4 days. 2. The two major drug-related components in the bile were identified as the glucuronide conjugates of 4-oxo-isotretinoin and 16-hydroxy-isotretinoin. Two minor components were identified as the glucuronide conjugates of isotretinoin and 18-hydroxy-isotretinoin. 3. H.p.l.c. analyses of Glusulase-treated bile samples indicated that the glucuronides of isotretinoin and the two major metabolites accounted for about 48% and 44% of the total radioactivity in the bile of the two patients. 4. Racemic 16-hydroxy-isotretinoin was synthesized and evaluated for its effect on human sebocytes in vitro. This metabolite and the other major metabolites of isotretinoin were less active than isotretinoin in inhibiting the proliferation of the sebocytes.