Oral pretreatment with β-lactoglobulin derived peptide and CpG co-encapsulated in PLGA nanoparticles prior to sensitizations attenuates cow's milk allergy development in mice

Cow's milk allergy is a common food allergy among infants. Improved hygiene conditions and loss of microbial diversity are associated with increased risk of allergy development. The intestinal immune system is essential for oral tolerance induction. In this respect, bacterial CpG DNA is known to drive Th1 and regulatory T-cell (Treg) development via Toll-Like-Receptor 9 (TLR-9) signaling, skewing away from the allergic Th2 phenotype. We aimed to induce allergen specific tolerance via oral delivery of poly (lactic-co-glycolic acid) nanoparticles (NP) co-encapsulated with a selected β-lactoglobulin derived peptide (BLG-Pep) and TLR-9 ligand CpG oligodeoxynucleotide (CpG). In vivo, 3-4-week-old female C3H/HeOuJ mice housed in individually ventilated cages received 6-consecutive-daily gavages of either PBS, whey, BLG-Pep/NP, CpG/NP, a mixture of BLG-Pep/NP plus CpG/NP or co-encapsulated BLG-Pep+CpG/NP, before 5-weekly oral sensitizations with whey plus cholera toxin (CT) or only CT (sham) and were challenged with whey 5 days after the last sensitization. The co-encapsulated BLG-Pep+CpG/NP pretreatment, but not BLG-Pep/NP, CpG/NP or the mixture of BLG-Pep/NP plus CpG/NP, prevented the whey-induced allergic skin reactivity and prevented rise in serum BLG-specific IgE compared to whey-sensitized mice. Importantly, co-encapsulated BLG-Pep+CpG/NP pretreatment reduced dendritic cell (DC) activation and lowered the frequencies of PD-L1+ DC in the mesenteric lymph nodes compared to whey-sensitized mice. By contrast, co-encapsulated BLG-Pep+CpG/NP pretreatment increased the frequency of splenic PD-L1+ DC compared to the BLG-Pep/NP plus CpG/NP recipients, in association with lower Th2 development and increased Treg/Th2 and Th1/Th2 ratios in the spleen. Oral administration of PLGA NP co-encapsulated with BLG-Pep and CpG prevented rise in serum BLG-specific IgE and symptom development while lowering splenic Th2 cell frequency in these mice which were kept under strict hygienic conditions

Oral pretreatment with β-lactoglobulin derived peptide and CpG co-encapsulated in PLGA nanoparticles prior to sensitizations attenuates cow’s milk allergy development in mice

Cow’s milk allergy is a common food allergy among infants. Improved hygiene conditions and loss of microbial diversity are associated with increased risk of allergy development. The intestinal immune system is essential for oral tolerance induction. In this respect, bacterial CpG DNA is known to drive Th1 and regulatory T-cell (Treg) development via Toll-Like-Receptor 9 (TLR-9) signaling, skewing away from the allergic Th2 phenotype. We aimed to induce allergen specific tolerance via oral delivery of poly (lactic-co-glycolic acid) nanoparticles (NP) co-encapsulated with a selected β-lactoglobulin derived peptide (BLG-Pep) and TLR-9 ligand CpG oligodeoxynucleotide (CpG). In vivo, 3-4-week-old female C3H/HeOuJ mice housed in individually ventilated cages received 6-consecutive-daily gavages of either PBS, whey, BLG-Pep/NP, CpG/NP, a mixture of BLG-Pep/NP plus CpG/NP or co-encapsulated BLG-Pep+CpG/NP, before 5-weekly oral sensitizations with whey plus cholera toxin (CT) or only CT (sham) and were challenged with whey 5 days after the last sensitization. The co-encapsulated BLG-Pep+CpG/NP pretreatment, but not BLG-Pep/NP, CpG/NP or the mixture of BLG-Pep/NP plus CpG/NP, prevented the whey-induced allergic skin reactivity and prevented rise in serum BLG-specific IgE compared to whey-sensitized mice. Importantly, co-encapsulated BLG-Pep+CpG/NP pretreatment reduced dendritic cell (DC) activation and lowered the frequencies of PD-L1+ DC in the mesenteric lymph nodes compared to whey-sensitized mice. By contrast, co-encapsulated BLG-Pep+CpG/NP pretreatment increased the frequency of splenic PD-L1+ DC compared to the BLG-Pep/NP plus CpG/NP recipients, in association with lower Th2 development and increased Treg/Th2 and Th1/Th2 ratios in the spleen. Oral administration of PLGA NP co-encapsulated with BLG-Pep and CpG prevented rise in serum BLG-specific IgE and symptom development while lowering splenic Th2 cell frequency in these mice which were kept under strict hygienic conditions

The Combination Therapy of Dietary Galacto-Oligosaccharides With Budesonide Reduces Pulmonary Th2 Driving Mediators and Mast Cell Degranulation in a Murine Model of House Dust Mite Induced Asthma

Background: Dietary non-digestible galacto-oligosaccharides (GOS) suppress allergic responses in mice sensitized and challenged with house dust mite (HDM). Budesonide is the standard therapy for allergic asthma in humans but is not always completely effective.Aim: To compare the efficacy of budesonide or different doses of GOS alone or with a combination therapy of budesonide and GOS on HDM-allergic responses in mice.Methods:BALB/c mice were sensitized and challenged with HDM, while fed a control diet or a diet supplemented with 1 or 2.5 w/w% GOS, and either or not oropharyngeally instilled with budesonide. Systemic and local inflammatory markers, such as mucosal mast cell protease-1 (mMCP-1) in serum, pulmonary CCL17, CCL22, and IL-33 concentrations and inflammatory cell influx in the bronchoalveolar lavage fluid (BALF) were determined.Results: Budesonide or GOS alone suppressed the number of eosinophils in the BALF of HDM allergic mice whereas budesonide either or not combined with GOS lowered both eosinophil and lymphocyte numbers in the BALF of HDM-allergic mice. Both 1 w/w% and 2.5 w/w% GOS and/or budesonide suppressed serum mMCP-1 concentrations. However, budesonide nor GOS alone was capable of reducing Th2 driving chemokines CCL17, CCL22 and IL-33 protein levels in supernatants of lung homogenates of HDM allergic mice, whereas the combination therapy did. Moreover, IL-13 concentrations were only significantly suppressed in mice treated with budesonide while fed GOS. A similar tendency was observed for the frequency of GATA3+CD4+ Th2 and CD4+RORγt+ Th17 cells in the lungs of the allergic mice.Conclusion: Dietary intervention using GOS may be a novel way to further improve the efficacy of anti-inflammatory drug therapy in allergic asthma by lowering Th2 driving mediators and mast cell degranulation

Galectin-1 and platelet factor 4 (CXCL4) induce complementary platelet responses in vitro

Galectin-1 (gal-1) is a carbohydrate-binding lectin with important functions in angiogenesis, immune response, hemostasis and inflammation. Comparable functions are exerted by platelet factor 4 (CXCL4), a chemokine stored in the α-granules of platelets. Previously, gal-1 was found to activate platelets through integrin αIIbβ3. Both gal-1 and CXCL4 have high affinities for polysaccharides, and thus may mutually influence their functions. The aim of this study was to investigate a possible synergism of gal-1 and CXCL4 in platelet activation. Platelets were treated with increasing concentrations of gal-1, CXCL4 or both, and aggregation, integrin activation, P-selectin and phosphatidyl serine (PS) exposure were determined by light transmission aggregometry and by flow cytometry. To investigate the influence of cell surface sialic acid, platelets were treated with neuraminidase prior to stimulation. Gal-1 and CXCL4 were found to colocalize on the platelet surface. Stimulation with gal-1 led to integrin αIIbβ3 activation and to robust platelet aggregation, while CXCL4 weakly triggered aggregation and primarily induced P-selectin expression. Co-incubation of gal-1 and CXCL4 potentiated platelet aggregation compared with gal-1 alone. Whereas neither gal-1 and CXCL4 induced PS-exposure on platelets, prior removal of surface sialic acid strongly potentiated PS exposure. In addition, neuraminidase treatment increased the binding of gal-1 to platelets and lowered the activation threshold for gal-1. However, CXCL4 did not affect binding of gal-1 to platelets. Taken together, stimulation of platelets with gal-1 and CXCL4 led to distinct and complementary activation profiles, with additive rather than synergistic effects

Ultrasound image analysis offers the opportunity to predict plasma progesterone concentrations in the estrous cycle in cows: a feasibility study

In recent years, several attempts have been made to evaluate the activity of a corpus luteum by determining its sonographic echo texture. In all of these studies the values of the echo texture parameters depended on the type and settings of the ultrasound machine. Therefore, the aim of the study was to investigate if a quantitative analysis of ultrasound (US) images of the corpus luteum (CL) after calibration of the ultrasound machine enables the assessment of the peripheral plasma progesterone (P4) level. Ten Holstein Friesian cows were examined daily at Days 4 to 8, 10 to 16, and -5 to -1 (Day 1=ovulation) of the estrous cycle. B-mode sonography of the corpora lutea was performed and blood samples were taken for plasma P4 analysis. US images were calibrated and analyzed using a software package (CAUS) developed by the authors. In addition to the area of the CL (Total Area, TotA; Tissue Area interactive, TisAi; Tissue Area Automatic, TisAa), the following US parameters were calculated from the gray level histogram and from the size of the speckles: Mean, Standard Deviation (SD) and Signal-to-Noise Ratio (SNR=Mean/SD) of echo levels, Residual Attenuation (ResAtt), Axial and Lateral speckle size (Ax and Lat, respectively). The inter-individual variability of the P4 level was expressed by the coefficient of variability (CV), averaged over all days. It appeared that the CV of the absolute P4 was high (0.65) and the P4 relative to that at Day 4 and at Day 16 was of comparable magnitude. Correlations of US parameters with P4 were highest for the P4 relative to Day 16 (P4_rel_D16). This relative P4 measure was then used for further analysis. The correlations of P4_rel_D16 with TotA, TisAa (CL area after automatic segmentation of tissue) and ResAtt were found the highest (R=0.68, 0.74, and -0.42, respectively). Multiple linear regression analysis, incorporating all US parameters revealed the formula: P4_rel_D16(pred)=-0.315+0.225TisAa-0.023ResAtt, and a goodness of fit: R(2)=0.59 (p0.80 (corresponding to 0.95 times the average_P4_rel_D16 measured during the "static" phase of the luteal cycle) by ROC analysis was correctly made in 88% of cases. In conclusion the quantitative analysis of calibrated ultrasound images may yield a good prediction of cyclic changes of P4 levels and has potential for predicting the phase in the estrous cycle of a cow

In vivo 3D cardiac and skeletal muscle strain estimation

In this study, BiPlane imaging was adapted for measuring strain in actively deforming tissue in three orthogonal directions. BiPlane imaging assures a sufficient frame rate (75-120 Hz) for accurate strain estimation. A coarse-to-fine iterative 2D strain algorithm using spatial correction and local stretching was implemented. Considering the huge amount of generated data, a fast interpolation scheme was implemented for measuring sub-sample and sub-line displacements. Assuming a 2D parabolic shape of the cross-correlation function, a straightforward and direct calculation of the displacements is possible. The strain estimation method was validated by means of a simulation study and phantom experiments. Rf-data were acquired with a 3D X4 matrix array transducer (Philips Sonos 7500) in BiPlane mode. In vivo verification in human skeletal muscle was performed. Furthermore, cardiac strain imaging was conducted using cardiac BiPlane data of dogs. In a pilot animal study, beagles with an induced valvular aortic stenosis were monitored. The Field II simulation was used for determining the accuracy and detectibility of the algorithm and revealed excellent correlation between applied and measured axial strain (SNR = 43 dB) for a window of 0.60 mm. Obviously, a lower SNR was found in lateral and elevational direction. The in vivo verification experiment in the skeletal muscles revealed similar cumulative axial strain curves (up to 8%) in both the azimuth and elevational direction. The shape of the strain curve matched perfectly with the curve of the measured force. The lateral strain values parallel to the direction of the muscle fibers matched the axial strain curves, whereas the shape of the lateral strain in the perpendicular plane differed due to anisotropy. Finally, strain images of the beagles were calculated. The beagle with the most excessive pressure gradient revealed a decrease of the radial strain. Furthermore, an elongated plateau in the radial strain indicated hypertrophy. In conclusion, 3D cardiac and strain estimation is feasible using a real-time 3D scanner. Additional validation studies of full 3D imaging modes are required to fully validate the technique

The Combination Therapy of Dietary Galacto-Oligosaccharides With Budesonide Reduces Pulmonary Th2 Driving Mediators and Mast Cell Degranulation in a Murine Model of House Dust Mite Induced Asthma

Background: Dietary non-digestible galacto-oligosaccharides (GOS) suppress allergic responses in mice sensitized and challenged with house dust mite (HDM). Budesonide is the standard therapy for allergic asthma in humans but is not always completely effective. Aim: To compare the efficacy of budesonide or different doses of GOS alone or with a combination therapy of budesonide and GOS on HDM-allergic responses in mice. Methods:BALB/c mice were sensitized and challenged with HDM, while fed a control diet or a diet supplemented with 1 or 2.5 w/w% GOS, and either or not oropharyngeally instilled with budesonide. Systemic and local inflammatory markers, such as mucosal mast cell protease-1 (mMCP-1) in serum, pulmonary CCL17, CCL22, and IL-33 concentrations and inflammatory cell influx in the bronchoalveolar lavage fluid (BALF) were determined. Results: Budesonide or GOS alone suppressed the number of eosinophils in the BALF of HDM allergic mice whereas budesonide either or not combined with GOS lowered both eosinophil and lymphocyte numbers in the BALF of HDM-allergic mice. Both 1 w/w% and 2.5 w/w% GOS and/or budesonide suppressed serum mMCP-1 concentrations. However, budesonide nor GOS alone was capable of reducing Th2 driving chemokines CCL17, CCL22 and IL-33 protein levels in supernatants of lung homogenates of HDM allergic mice, whereas the combination therapy did. Moreover, IL-13 concentrations were only significantly suppressed in mice treated with budesonide while fed GOS. A similar tendency was observed for the frequency of GATA3+CD4+ Th2 and CD4+RORγt+ Th17 cells in the lungs of the allergic mice. Conclusion: Dietary intervention using GOS may be a novel way to further improve the efficacy of anti-inflammatory drug therapy in allergic asthma by lowering Th2 driving mediators and mast cell degranulation

Photoacoustic determination of blood vessel diameter

A double-ring sensor was applied in photoacoustic tomographic imaging of artificial blood vessels as well as blood vessels in a rabbit ear. The peak-topeak time (τ pp) of the laser (1064 nm) induced pressure transient was used to estimate the axial vessel diameter. Comparison with the actual vessel diameter showed that the diameter could be approximated by 2cτ pp, with c the speed of sound in blood. Using this relation, the lateral diameter could also precisely be determined. In vivo imaging and monitoring of changes in vessel diameters was feasible. Finally, acoustic time traces were recorded while flushing a vessel in the rabbit ear with saline, which proved that the main contribution to the laser-induced pressure transient is caused by blood inside the vessel and that the vessel wall gives only a minor contribution

Interactive vs. automatic ultrasound image segmentation methods for staging hepatic lipidosis.

Item does not contain fulltextThe aim of this study was to test the hypothesis that automatic segmentation of vessels in ultrasound (US) images can produce similar or better results in grading fatty livers than interactive segmentation. A study was performed in postpartum dairy cows (N=151), as an animal model of human fatty liver disease, to test this hypothesis. Five transcutaneous and five intraoperative US liver images were acquired in each animal and a liverbiopsy was taken. In liver tissue samples, triacylglycerol (TAG) was measured by biochemical analysis and hepatic diseases other than hepatic lipidosis were excluded by histopathologic examination. Ultrasonic tissue characterization (UTC) parameters--Mean echo level, standard deviation (SD) of echo level, signal-to-noise ratio (SNR), residual attenuation coefficient (ResAtt) and axial and lateral speckle size--were derived using a computer-aided US (CAUS) protocol and software package. First, the liver tissue was interactively segmented by two observers. With increasing fat content, fewer hepatic vessels were visible in the ultrasound images and, therefore, a smaller proportion of the liver needed to be excluded from these images. Automatic-segmentation algorithms were implemented and it was investigated whether better results could be achieved than with the subjective and time-consuming interactive-segmentation procedure. The automatic-segmentation algorithms were based on both fixed and adaptive thresholding techniques in combination with a 'speckle'-shaped moving-window exclusion technique. All data were analyzed with and without postprocessing as contained in CAUS and with different automated-segmentation techniques. This enabled us to study the effect of the applied postprocessing steps on single and multiple linear regressions ofthe various UTC parameters with TAG. Improved correlations for all US parameters were found by using automatic-segmentation techniques. Stepwise multiple linear-regression formulas where derived and used to predict TAG level in the liver. Receiver-operating-characteristics (ROC) analysis was applied to assess the performance and area under the curve (AUC) of predicting TAG and to compare the sensitivity and specificity of the methods. Best speckle-size estimates and overall performance (R2 = 0.71, AUC = 0.94) were achieved by using an SNR-based adaptive automatic-segmentation method (used TAG threshold: 50 mg/g liver wet weight). Automatic segmentation is thus feasible and profitable.1 juli 201