Allergen-specific antibody and cytokine responses, mast cell reactivity and intestinal permeability upon oral challenge of sensitized and tolerized mice.

BACKGROUND: Food allergy has reached an epidemic level in westernized countries and although central mechanisms have been described, the variability associated with genetic diversity underscores the still unresolved complexity of these disorders. OBJECTIVE: To develop models of food allergy and oral tolerance, both strictly induced by the intestinal route, and to compare antigen-specific responses. METHODS: BALB/c mice were mucosally sensitized to ovalbumin (OVA) in the presence of the mucosal adjuvant cholera toxin, or tolerized by intra-gastric administrations of OVA alone. Antibody titres and cytokines were determined by ELISA, and allergic status was determined through several physiologic parameters including decline in temperature, diarrhoea, mast cell degranulation and intestinal permeability. RESULTS: OVA-specific antibodies (IgE, IgGs and IgA in serum and feces) were produced in sensitized mice exclusively. Upon intra-gastric challenge with OVA, sensitized mice developed anaphylactic reactions associated with a decline of temperature, diarrhoea, degranulation of mast cells, which were only moderately recruited in the small intestine, and increased intestinal permeability. Cytokines produced by immune cells from sensitized mice included T-helper type 2 cytokines (IL-5, IL-13), but also IL-10, IFN-gamma and IL-17. In contrast, all markers of allergy were totally absent in tolerized animals, and yet the latter were protected from subsequent sensitization, demonstrating that oral tolerance took place efficiently. CONCLUSION: This work allows for the first time an appropriate comparison between sensitized and tolerized BALB/c mice towards OVA. It highlights important differences from other models of allergy, and thus questions some of the generally accepted notions of allergic reactions, such as the protective role of IFN-gamma, the importance of antigen-specific secretory IgA and the role of mucosal mast cells in intestinal anaphylaxis. In addition, it suggests that IL-17 might be an effector cytokine in food allergy. Finally, it demonstrates that intestinal permeability towards the allergen is increased during challenge.

Renal sodium handling in acute and chronic salt loading/depletion protocols: the confounding influence of acute water loading.

OBJECTIVES: Renal tubular sodium handling was measured in healthy subjects submitted to acute and chronic salt-repletion/salt-depletion protocols. The goal was to compare the changes in proximal and distal sodium handling induced by the two procedures using the lithium clearance technique. METHODS: In nine subjects, acute salt loading was obtained with a 2 h infusion of isotonic saline, and salt depletion was induced with a low-salt diet and furosemide. In the chronic protocol, 15 subjects randomly received a low-, a regular- and a high-sodium diet for 1 week. In both protocols, renal and systemic haemodynamics and urinary electrolyte excretion were measured after an acute water load. In the chronic study, sodium handling was also determined, based on 12 h day- and night-time urine collections. RESULTS: The acute and chronic protocols induced comparable changes in sodium excretion, renal haemodynamics and hormonal responses. Yet, the relative contribution of the proximal and distal nephrons to sodium excretion in response to salt loading and depletion differed in the two protocols. Acutely, subjects appeared to regulate sodium balance mainly by the distal nephron, with little contribution of the proximal tubule. In contrast, in the chronic protocol, changes in sodium reabsorption could be measured both in the proximal and distal nephrons. Acute water loading was an important confounding factor which increased sodium excretion by reducing proximal sodium reabsorption. This interference of water was particularly marked in salt-depleted subjects. CONCLUSION: Acute and chronic salt loading/salt depletion protocols investigate different renal mechanisms of control of sodium balance. The endogenous lithium clearance technique is a reliable method to assess proximal sodium reabsorption in humans. However, to investigate sodium handling in diseases such as hypertension, lithium should be measured preferably on 24 h or overnight urine collections to avoid the confounding influence of water.

Technology transfer of an oil-in-water vaccine-adjuvant for strengthening pandemic influenza preparedness in Indonesia.

With the current enzootic circulation of highly pathogenic avian influenza viruses, the ability to increase global pandemic influenza vaccine production capacity is of paramount importance. This has been highlighted by, and is one of the main pillars of, the WHO Global Action Plan for Influenza Vaccines (GAP). Such capacity expansion is especially relevant in developing countries. The Vaccine Formulation Laboratory at University of Lausanne is engaged in the technology transfer of an antigen-sparing oil-in-water adjuvant in order to empower developing countries vaccine manufacturers to increase pandemic influenza vaccine capacity. In a one-year project funded by United States Department of Health and Human Services, the Vaccine Formulation Laboratory transferred the process know-how and associated equipment for the pilot-scale manufacturing of an oil-in-water adjuvant to Bio Farma, Indonesia's state-owned vaccine manufacturer, for subsequent formulation with H5N1 pandemic influenza vaccines. This paper describes the experience acquired and lessons learnt from this technology transfer project.

Pathogen-induced hatching and population-specific life-history response to water-borne cues in brown trout (Salmo trutta)

Hatching is an important niche shift, and embryos in a wide range of taxa can either accelerate or delay this life-history switch in order to avoid stage-specific risks. Such behavior can occur in response to stress itself and to chemical cues that allow anticipation of stress. We studied the genetic organization of this phenotypic plasticity and tested whether there are differences among populations and across environments in order to learn more about the evolutionary potential of stress-induced hatching. As a study species, we chose the brown trout (Salmo trutta; Salmonidae). Gametes were collected from five natural populations (within one river network) and used for full-factorial in vitro fertilizations. The resulting embryos were either directly infected with Pseudomonas fluorescens or were exposed to waterborne cues from P. fluorescens-infected conspecifics. We found that direct inoculation with P. fluorescens increased embryonic mortality and induced hatching in all host populations. Exposure to waterborne cues revealed population-specific responses. We found significant additive genetic variation for hatching time, and genetic variation in trait plasticity. In conclusion, hatching is induced in response to infection and can be affected by waterborne cues of infection, but populations and families differ in their reaction to the latter.

Investigating the Effect of Aloe vera Gel on the Buccal Permeability of Didanosine.

The buccal mucosal route offers several advantages but the delivery of certain drugs can be limited by low membrane permeability. This study investigated the buccal permeability properties of didanosine (ddI) and assessed the potential of ALOE VERA gel (AVgel) as a novel buccal permeation enhancer. Permeation studies were performed using Franz diffusion cells, and the drug was quantified by UV spectroscopy. Histomorphological evaluations were undertaken using light and transmission electron microscopy. The permeability of ddI was concentration-dependent, and it did not have any adverse effects on the buccal mucosae. A linear relationship (R (2) = 0.9557) between the concentrations and flux indicated passive diffusion as the mechanism of drug transport. AVgel at concentrations of 0.25 to 2 %w/v enhanced ddI permeability with enhancement ratios from 5.09 (0.25 %w/v) to 11.78 (2 %w/v) but decreased permeability at 4 and 6 %w/v. Ultrastructural analysis of the buccal mucosae treated with phosphate buffer saline pH 7.4 (PBS), ddI/PBS, and ddI/PBS/AVgel 0.5 %w/v showed cells with normal plasmalemma, well-developed cristae, and nuclei with regular nuclear envelopes. However, cells from 1, 2, and 6 %w/v AVgel-treated mucosae showed irregular nuclear outlines, increased intercellular spacing, and plasmalemma crenulations. This study demonstrates the potential of AVgel as a buccal permeation enhancer for ddI to improve anti-HIV and AIDS therapy.

A double layer model of the gas bubble/water interface

Zeta potential is a physico-chemical parameter of particular importance to describe sorption of contaminants at the surface of gas bubbles. Nevertheless, the interpretation of electrophoretic mobilities of gas bubbles is complex. This is due to the specific behavior of the gas at interface and to the excess of electrical charge at interface, which is responsible for surface conductivity. We developed a surface complexation model based on the presence of negative surface sites because the balance of accepting and donating hydrogen bonds is broken at interface. By considering protons adsorbed on these sites followed by a diffuse layer, the electrical potential at the head-end of the diffuse layer is computed and considered to be equal to the zeta potential. The predicted zeta potential values are in very good agreement with the experimental data of H-2 bubbles for a broad range of pH and NaCl concentrations. This implies that the shear plane is located at the head-end of the diffuse layer, contradicting the assumption of the presence of a stagnant diffuse layer at the gas/water interface. Our model also successfully predicts the surface tension of air bubbles in a KCl solution. (c) 2012 Elsevier Inc. All rights reserved.

Determination of Transmembrane Water Fluxes in Neurons Elicited by Glutamate Ionotropic Receptors and by the Cotransporters KCC2 and NKCC1: A Digital Holographic Microscopy Study.

Digital holographic microscopy (DHM) is a noninvasive optical imaging technique that provides quantitative phase images of living cells. In a recent study, we showed that the quantitative monitoring of the phase signal by DHM was a simple label-free method to study the effects of glutamate on neuronal optical responses (Pavillon et al., 2010). Here, we refine these observations and show that glutamate produces the following three distinct optical responses in mouse primary cortical neurons in culture, predominantly mediated by NMDA receptors: biphasic, reversible decrease (RD) and irreversible decrease (ID) responses. The shape and amplitude of the optical signal were not associated with a particular cellular phenotype but reflected the physiopathological status of neurons linked to the degree of NMDA activity. Thus, the biphasic, RD, and ID responses indicated, respectively, a low-level, a high-level, and an "excitotoxic" level of NMDA activation. Moreover, furosemide and bumetanide, two inhibitors of sodium-coupled and/or potassium-coupled chloride movement strongly modified the phase shift, suggesting an involvement of two neuronal cotransporters, NKCC1 (Na-K-Cl) and KCC2 (K-Cl) in the genesis of the optical signal. This observation is of particular interest since it shows that DHM is the first imaging technique able to monitor dynamically and in situ the activity of these cotransporters during physiological and/or pathological neuronal conditions.

In vivo characterisation of a novel water-soluble Cyclosporine A prodrug for the treatment of dry eye disease.

Cyclosporine A (CsA) has been demonstrated to be effective for the treatment of a variety of ophthalmological conditions, including ocular surface disorders such as the dry eye disease (DED). Since CsA is characterised by its low water solubility, the development of a topical ophthalmic formulation represents an interesting pharmaceutical question. In the present study, two different strategies to address this challenge were studied and compared: (i) a water-soluble CsA prodrug formulated within an aqueous solution and (ii) a CsA oil-in-water emulsion (Restasis, Allergan Inc., Irvine, CA). First, the prodrug formulation was shown to have an excellent ocular tolerance as well as no influence on the basal tear production; maintaining the ocular surface properties remained unchanged. Then, in order to allow in vivo investigations, a specific analytical method based on ultra high pressure liquid chromatography coupled with triple quadrupole mass spectrometer (UHPLC-MS/MS) was developed and optimised to quantify CsA in ocular tissues and fluids. The CsA ocular kinetics in lachrymal fluid for both formulations were found to be similar between 15 min and 48 h. The CsA ocular distribution study evidenced the ability of the prodrug formulation to penetrate into the eye, achieving therapeutically active CsA levels in tissues of both the anterior and posterior segments. In addition, the detailed analysis of the in vivo data using a bicompartmental model pointed out a higher bioavailability and lower elimination rate for CsA when it is generated from the prodrug than after direct application as an emulsion. The interesting in vivo properties displayed by the prodrug solution make it a safe and suitable option for the treatment of DED.

Quantification of myocardial blood flow with 82Rb positron emission tomography: clinical validation with 15O-water.

PURPOSE: Quantification of myocardial blood flow (MBF) with generator-produced (82)Rb is an attractive alternative for centres without an on-site cyclotron. Our aim was to validate (82)Rb-measured MBF in relation to that measured using (15)O-water, as a tracer 100% of which can be extracted from the circulation even at high flow rates, in healthy control subject and patients with mild coronary artery disease (CAD). METHODS: MBF was measured at rest and during adenosine-induced hyperaemia with (82)Rb and (15)O-water PET in 33 participants (22 control subjects, aged 30 ± 13 years; 11 CAD patients without transmural infarction, aged 60 ± 13 years). A one-tissue compartment (82)Rb model with ventricular spillover correction was used. The (82)Rb flow-dependent extraction rate was derived from (15)O-water measurements in a subset of 11 control subjects. Myocardial flow reserve (MFR) was defined as the hyperaemic/rest MBF. Pearson's correlation r, Bland-Altman 95% limits of agreement (LoA), and Lin's concordance correlation ρ (c) (measuring both precision and accuracy) were used. RESULTS: Over the entire MBF range (0.66-4.7 ml/min/g), concordance was excellent for MBF (r = 0.90, [(82)Rb-(15)O-water] mean difference ± SD = 0.04 ± 0.66 ml/min/g, LoA = -1.26 to 1.33 ml/min/g, ρ(c) = 0.88) and MFR (range 1.79-5.81, r = 0.83, mean difference = 0.14 ± 0.58, LoA = -0.99 to 1.28, ρ(c) = 0.82). Hyperaemic MBF was reduced in CAD patients compared with the subset of 11 control subjects (2.53 ± 0.74 vs. 3.62 ± 0.68 ml/min/g, p = 0.002, for (15)O-water; 2.53 ± 1.01 vs. 3.82 ± 1.21 ml/min/g, p = 0.013, for (82)Rb) and this was paralleled by a lower MFR (2.65 ± 0.62 vs. 3.79 ± 0.98, p = 0.004, for (15)O-water; 2.85 ± 0.91 vs. 3.88 ± 0.91, p = 0.012, for (82)Rb). Myocardial perfusion was homogeneous in 1,114 of 1,122 segments (99.3%) and there were no differences in MBF among the coronary artery territories (p > 0.31). CONCLUSION: Quantification of MBF with (82)Rb with a newly derived correction for the nonlinear extraction function was validated against MBF measured using (15)O-water in control subjects and patients with mild CAD, where it was found to be accurate at high flow rates. (82)Rb-derived MBF estimates seem robust for clinical research, advancing a step further towards its implementation in clinical routine.

Chemistry, isotope values (δD, δ18O, δ34S(SO4)) and temperatures of the water inflows in two Gotthard tunnels, Swiss Alps

Water inflows in the Gotthard Highway Tunnel and in the Gotthard Exploration Tunnel are meteoric waters infiltrating at different elevations, on both sides of an important orographic divide. Limited interaction of meteoric waters with gneissic rocks produces Ca-HCO3 and Na-Ca-HCO3 waters, whereas prolonged interaction of meteoric waters with the same rocks generates Na-HCO3 to Na-SO4 waters. Waters circulating in Triassic carbonate-evaporite rocks have a Ca-SO4 composition. Calcium-Na-SO4 waters are also present. They can be produced through interaction of either Na-HCO3 waters with anhydrite or Ca-SO4 waters with a local gneissic rock, as suggested by reaction path modeling. An analogous simulation indicates that Na-HCO3 waters are generated through interaction of Ca-HCO3 waters with a local gneissic rock. The two main SO4-sources present in the Alps are leaching of upper Triassic sulfate minerals and oxidative dissolution of sulfide minerals of crystalline rocks. Values of delta S-34(SO4) < <similar to>+ 9 parts per thousand, are due to oxidative dissolution of sulfide minerals, whereas delta S-34(SO4) > similar to+ 9 parts per thousand are controlled either by bacterial SO4 reduction or leaching of upper Triassic sulfate minerals. Most waters have temperatures similar to the expected values for a geothermal gradient of 22 degreesC/km and are close to thermal equilibrium with rocks. However relatively large, descending flows of cold waters and ascending flows of warm waters are present in both tunnels and determine substantial cooling and heating, respectively, of the interacting rocks. The most import upflow zone of warm, Na-rich waters is below Guspisbach, in the Gotthard Highway Tunnel, at 6.2-9.0 km from the southern portal. These warm waters have equilibrium temperatures of 65-75 degreesC and therefore constitute an important low-enthalpy geothermal resource. (C) 2001 Elsevier Science Ltd. All rights reserved.

Fat signal suppression for coronary MRA at 3T using a water-selective adiabatic T2 -preparation technique.

PURPOSE: To improve fat saturation in coronary MRA at 3T by using a spectrally selective adiabatic T2 -Prep (WSA-T2 -Prep). METHODS: A conventional adiabatic T2 -Prep (CA-T2 -Prep) was modified, such that the excitation and restoration pulses were of differing bandwidths. On-resonance spins are T2 -Prepared, whereas off-resonance spins, such as fat, are spoiled. This approach was combined with a CHEmically Selective Saturation (CHESS) pulse to achieve even greater fat suppression. Numerical simulations were followed by phantom validation and in vivo coronary MRA. RESULTS: Numerical simulations demonstrated that augmenting a CHESS pulse with a WSA-T2 -Prep improved robustness to B1 inhomogeneities and that this combined fat suppression was effective over a broader spectral range than that of a CHESS pulse in a conventional T2 -Prepared sequence. Phantom studies also demonstrated that the WSA-T2 -Prep+CHESS combination produced greater fat suppression across a range of B1 values than did a CA-T2 -Prep+CHESS combination. Lastly, in vivo measurements demonstrated that the contrast-to-noise ratio between blood and myocardium was not adversely affected by using a WSA-T2 -Prep, despite the improved abdominal and epicardial fat suppression. Additionally, vessel sharpness improved. CONCLUSION: The proposed WSA-T2 -Prep method was shown to improve fat suppression and vessel sharpness as compared to a CA-T2 -Prep technique, and to also increase fat suppression when combined with a CHESS pulse.

Peroxisome-proliferator-activated receptor (PPAR)-gamma activation stimulates keratinocyte differentiation.

Previous studies demonstrated that peroxisome-proliferator-activated receptor (PPAR)-alpha or PPAR-delta activation stimulates keratinocyte differentiation, is anti-inflammatory, and improves barrier homeostasis. Here we demonstrate that treatment of cultured human keratinocytes with ciglitazone, a PPAR-gamma activator, increases involucrin and transglutaminase 1 mRNA levels. Moreover, topical treatment of hairless mice with ciglitazone or troglitazone increases loricrin, involucrin, and filaggrin expression without altering epidermal morphology. These results indicate that PPAR-gamma activation stimulates keratinocyte differentiation. Additionally, PPAR-gamma activators accelerated barrier recovery following acute disruption by either tape stripping or acetone treatment, indicating an improvement in permeability barrier homeostasis. Treatment with PPAR-gamma activators also reduced the cutaneous inflammatory response that is induced by phorbol 12-myristate-13-acetate, a model of irritant contact dermatitis and oxazolone, a model of allergic contact dermatitis. To determine whether the effects of PPAR-gamma activators are mediated by PPAR-gamma, we next examined animals deficient in PPAR-gamma. Mice with a deficiency of PPAR-gamma specifically localized to the epidermis did not display any cutaneous abnormalites on inspection, but on light microscopy there was a modest increase in epidermal thickness associated with an increase in proliferating cell nuclear antigen (PCNA) staining. Key functions of the skin including permeability barrier homeostasis, stratum corneum surface pH, and water-holding capacity, and response to inflammatory stimuli were not altered in PPAR-gamma-deficient epidermis. Although PPAR-gamma activators stimulated loricrin and filaggrin expression in wild-type animals, however, in PPAR-gamma-deficient mice no effect was observed indicating that the stimulation of differentiation by PPAR-gamma activators is mediated by PPAR-gamma. In contrast, PPAR-gamma activators inhibited inflammation in both PPAR-gamma-deficient and wild-type mouse skin, indicating that the inhibition of cutaneous inflammation by these PPAR-gamma activators does not require PPAR-gamma in keratinocytes. These observations suggest that thiazolidindiones and perhaps other PPAR-gamma activators maybe useful in the treatment of cutaneous disorders.