In silico and in vivo protective effect of Morus nigra leaves on oxidative damage induced by iron overload.

Morus nigra L. is a plant popularly known as 'amoreira preta', very used in folk medicine. Iron overload (hemochromatosis) is a clinical condition that causes damage to various tissues due to oxidative stress. Therapy to control iron overload is still unsatisfactory. The protective effect on oxidative stress induced by iron overload was verified. Phytochemical characterization was evaluated by UHPLC-MS/MS. The in silico toxicity predictions of the main phytochemicals were performed via computer simulation. To induce iron overload, the animals received iron dextran (50 mg/kg/day). The test groups received doses of 500 and 1000 mg/kg of M. nigra extract for six weeks. Body weight, organosomatic index, serum iron, hepatic markers, cytokines, interfering factors in iron metabolism, enzymatic and histopathological evaluations were analyzed. Vanillic acid, caffeic acid, 6-hydroxycoumarin, p-coumaric acid, ferulic acid, rutin, quercitrin, resveratrol, apigenin and kaempferol were identified in the extract. In addition, in silico toxic predictions showed that the main compounds presented a low probability of toxic risk. The extract of M. nigra showed to control the mediators of inflammation and to reduce iron overload in several tissues. Our findings illustrate a novel therapeutic action of M. nigra leaves on hemochromatosis caused by iron overload.

Effect of Iontophoresis on the Intradermal Migration Rate of Medium Molecular Weight Drugs.

The purpose of the present study was to evaluate whether iontophoresis (IP) accelerates the intradermal migration rate of medium molecular weight drugs. Sodium polystyrene sulfonate (PSA) and fluorescein isothiocyanate-dextran (FD) were used as model medium molecular weight acidic and non-electrolyte drugs, respectively. Low molecular weight acid and non-electrolyte drugs were also used for comparison. Drug-loaded excised split-layered skin (SL skin) was used in the experiment. SL skin was prepared using (i) whole skin was split once, (ii) the drug solution was applied on the lower skin, and (iii) the upper skin was layered onto the lower skin containing the drug solution as in the original skin. The effect of constant-current cathodal or anodal IP was applied to the SL skin, and the time course of the cumulative amount of drug migration from the SL skin through the dermis to the receiver was followed. In cases without IP and with anodal IP, the intradermal migration rates of medium molecular weight drugs were much lower than those of small molecules. The driving force for drug migration was thought to be simple diffusion through the skin layer. In contrast, cathodal IP significantly increased the intradermal migration rate of PSA not but of FD or low molecular weight drugs. This IP-facilitated migration of PSA was probably due to electrorepulsion. These results suggest that IP can be used to increase the intradermal migration of medium molecular weight charged drugs.

Evaluation of anti-biofouling potential of Viburnum opulus extracts / Evaluación del potencial anti bioincrustante de extractos de Viburnum opulus

In this study the in vitro investigation of the inhibitory effect of ethanol extract of Viburnum opulus L. bark sample on Streptococcus mutans planctonic cells and biofilm has been intended. A Scanning electron microscopy analysis has been performed in order to investigate the inhibitory effect of the extract on Streptococcus mutans biofilms. Furthermore, the Exopolysaccharide and dextran production of this bacteria have been identified in the presence of the extract. It has been found out that the bark extract with the concentration of 2,5 mg/mL is able to inhibit more than 50% of the cells in the different times development phases. According to this, the exopolymeric matrix on the biofilm surface disperses and the Exopolysaccharide and dextran production get lowered in the presence of bark extract compared to the control group. It is considered that this extract can be used as an alternative approach for the new chemotherapeutic strategies against tooth decay.

En este estudio se investigó el efecto inhibitorio in vitro del extracto de etanólico de una muestra de corteza de Viburnum opulus L. en biopelículas de células planctónicas de Streptococcus mutans. Se realizó un análisis de microscopía electrónica de barrido para investigar el efecto inhibitorio del extracto sobre las biopelículas de Streptococcus mutans. Además, se identificó la producción de exopolisacárido y dextrano de esta bacteria en presencia del extracto. Se descubrió que el extracto de corteza con una concentración de 2,5 mg/ml inhibió más del 50% de las células en las diferentes fases de desarrollo. Consecuentemente, la matriz exopolimérica en la superficie de la biopelícula se dispersa y la producción de exopolisacárido y dextrano se reduce en presencia de extracto de corteza en comparación con el grupo de control. Se sugiere que este extracto puede ser usado como un enfoque alternativo para las nuevas estrategias quimioterapéuticas contra la carie dental.

Ubiquitous macropinocytosis in anthozoans.

Transport of fluids, molecules, nutrients or nanoparticles through coral tissues are poorly documented. Here, we followed the flow of various tracers from the external seawater to within the cells of all tissues in living animals. After entering the general coelenteric cavity, we show that nanoparticles disperse throughout the tissues via the paracellular pathway. Then, the ubiquitous entry gate to within the cells' cytoplasm is macropinocytosis. Most cells form large vesicles of 350-600 nm in diameter at their apical side, continuously internalizing their surrounding medium. Macropinocytosis was confirmed using specific inhibitors of PI3K and actin polymerization. Nanoparticle internalization dynamics is size dependent and differs between tissues. Furthermore, we reveal that macropinocytosis is likely a major endocytic pathway in other anthozoan species. The fact that nearly all cells of an animal are continuously soaking in the environment challenges many aspects of the classical physiology viewpoints acquired from the study of bilaterians.

Biodistribution and toxicity of epitope-functionalized dextran iron oxide nanoparticles in a pregnant murine model.

In pursuit of a preventive therapeutic for maternal autoantibody-related (MAR) autism, we assessed the toxicity, biodistribution, and clearance of a MAR specific peptide-functionalized dextran iron oxide nanoparticle system in pregnant murine dams. We previously synthesized ~15 nm citrate-coated dextran iron oxide nanoparticles (DIONPs), surface-modified with polyethylene glycol and MAR peptides to produce systems for nanoparticle-based autoantibody reception and entrapments (SNAREs). First, we investigated their immunogenicity and MAR lactate dehydrogenase B antibody uptake in murine serum in vitro. To assess biodistribution and toxicity, as well as systemic effects, we performed in vivo clinical and post mortem pathological evaluations. We observed minimal production of inflammatory cytokines-interleukin 10 (IL-10) and IL-12 following in vitro exposure of macrophages to SNAREs. We established the maximum tolerated dose of SNAREs to be 150 mg/kg at which deposition of iron was evident in the liver and lungs by histology and magnetic resonance imaging but no concurrent evidence of liver toxicity or lung infarction was detected. Further, SNAREs exhibited slower clearance from the maternal blood in pregnant dams compared to DIONPs based on serum total iron concentration. These findings demonstrated that the SNAREs have a prolonged presence in the blood and are safe for use in pregnant mice as evidenced by no associated organ damage, failure, inflammation, and fetal mortality. Determination of the MTD dose sets the basis for future studies investigating the efficacy of our nanoparticle formulation in a MAR autism mouse model.

Radiation Inhibits Lymph Drainage in an Acquired Lymphedema Mouse Hindlimb Model.

Background: Radiation therapy has been applied to prolong the duration of lymphedema. This study aimed to evaluate the effect of radiation on the development of lymphedema in a mouse hindlimb model. Methods and Results: A total of 24 Balb/c mice underwent the right popliteal lymph node excision and the afferent and efferent lymphatics blockage. The radiation group (n = 12) received a single 20 Gy radiation 1 day before surgery in the right hindlimb of each mouse, whereas the control group (n = 12) only received surgery without radiation. The right hindpaw thickness of each mouse was measured twice a week for 4 weeks. Fluorescence microscopy images using fluorescein isothiocyanate-dextran tracer were obtained once weekly. Immunohistochemical (IHC) staining images using anti-lymphatic vessel endothelial hyaluronan receptor-1 (anti-LYVE-1) were obtained at 4 weeks after surgery. The radiation group showed significant increase in the thickness of the right hind paws from 0.5 to 2 weeks compared with the control group. As for fluorescence lymphography, the radiation group showed a lower number of regenerated lymphatics and more congestion of tracers in the operated limb at the surgery sites at 1, 2, 3, and 4 weeks after surgery. For the IHC analysis, the radiation group showed a lower number of regenerated lymphatics per high-power field at the surgery site than the control group. Conclusion: Radiation therapy transiently aggravated the extent of lymphedema by inhibiting regenerated lymphatics in a mouse hindlimb model. However, it did not prolong the duration of lymphedema because the cutaneous interstitial flow contributes to the lymphatic fluid clearance.

Single microcolony diffusion analysis in Pseudomonas aeruginosa biofilms.

The influence of the biofilm matrix on molecular diffusion is commonly hypothesized to be responsible for emergent characteristics of biofilms such as nutrient trapping, signal accumulation and antibiotic tolerance. Hence quantifying the molecular diffusion coefficient is important to determine whether there is an influence of biofilm microenvironment on the mobility of molecules. Here, we use single plane illumination microscopy fluorescence correlation spectroscopy (SPIM-FCS) to obtain 3D diffusion coefficient maps with micrometre spatial and millisecond temporal resolution of entire Pseudomonas aeruginosa microcolonies. We probed how molecular properties such as size and charge as well as biofilm properties such as microcolony size and depth influence diffusion of fluorescently labelled dextrans inside biofilms. The 2 MDa dextran showed uneven penetration and a reduction in diffusion coefficient suggesting that the biofilm acts as a molecular sieve. Its diffusion coefficient was negatively correlated with the size of the microcolony. Positively charged dextran molecules and positively charged antibiotic tobramycin preferentially partitioned into the biofilm and remained mobile inside the microcolony, albeit with a reduced diffusion coefficient. Lastly, we measured changes of diffusion upon induction of dispersal and detected an increase in diffusion coefficient inside the biofilm before any loss of biomass. Thus, the change in diffusion is a proxy to detect early stages of dispersal. Our work shows that 3D diffusion maps are very sensitive to physiological changes in biofilms, viz. dispersal. However, this study also shows that diffusion, as mediated by the biofilm matrix, does not account for the high level of antibiotic tolerance associated with biofilms.

Dual-modality imaging of atherosclerotic plaques using ultrasmall superparamagnetic iron oxide labeled with rhodamine.

Aim: The diagnosis of vulnerable atherosclerotic plaques remains challenging. This study labeled ultrasmall superparamagnetic iron oxide with rhodamine (USPIO-R) and evaluated USPIO-R for imaging atherosclerotic plaques. Methods: Apolipoprotein E-deficient mice were fed a high-fat diet and underwent MRI before and after an intravenous injection of USPIO-R. Subsequently, an aortic specimen from the mice was removed and sliced for fluorescence imaging and Prussian blue and immunofluorescent staining. Results: T2 signal loss appeared and persisted in the aortic plaque postinjection, and spontaneous fluorescence from the plaque was observed. The accumulated mechanism of USPIO-R by plaque was the macrophage internalization by Prussian blue and immunofluorescence. Conclusion: USPIO-R is a promising dual-modality probe for diagnosing and monitoring vulnerable atherosclerotic plaques.

MR and PET-CT monitoring of tissue-engineered vascular grafts in the ovine carotid artery.

The modification of biomaterials to comply with clinically employed monitoring techniques is a promising strategy to support clinical translation in regenerative medicine. Here, multimodal imaging of tissue-engineered vascular grafts (TEVG) was enabled by functionalizing the textile scaffold with ultrasmall superparamagnetic iron oxide (USPIO) nanoparticles. The resulting MR-imageable grafts (iTEVG) were monitored non-invasively throughout their whole life-cycle, from initial quality control to longitudinal functional evaluation in an ovine model for up to 8 weeks. Crucial features such as the complete embedding of the textile mesh in the developing tissue and the grafts' structural stability were assessed in vitro using 1T-, 3T- and 7T-MRI scanners. In vivo, the grafts were imaged by 3T-MRI and PET-CT. Contrary to unlabeled constructs, iTEVG could be delineated from native arteries and precisely localized by MRI. USPIO labeling neither induced calcifications, nor negatively affected their remodeling with respect to tissue-specific extracellular matrix composition and endothelialization. Functionality was confirmed by MR-angiography. 18F-FDG uptake (assessed via PET-CT) indicated only transient post-surgical inflammation. In conclusion, USPIO-labeling enables accurate localization of TEVG and opens up opportunities for multimodal imaging approaches to assess transplant acceptance and function. Thereby, it can support clinical decision-making on the need for further pharmacological or surgical interventions.

Capacitive Saccharide Sensor Based on Immobilized Phenylboronic Acid with Diol Specificity.

A capacitive sensor for saccharide detection is described in this study. The detection is based on selective interaction between diols and aminophenylboronic acid (APBA) immobilized on a gold electrode. Glucose, fructose, and dextran (MW: 40 kDa) were tested with the system over wide concentration ranges (1.0 x 10-8 M - 1.0 x 10-3 M for glucose, 1.0 x 10-8 M - 1.0 x 10-2 M for fructose and 1.0 x 10-10 M - 1.0 x 10-5 M for dextran). The limits of detection (LODs) were 0.8 nM for glucose, 0.6 nM for fructose, and 13 pM for dextran. These data were comparable to the others reported previously. In order to demonstrate glycoprotein detection with the same sensor, human immunoglobulin G (IgG) as well as horseradish peroxidase were used as model analytes. The sensor responded to IgG in the concentration range of 1.0 x 10-13 M - 1.0 x 10-7 M with a LOD value of 16 fM. The performance of the assay of peroxidase was compared to a spectrophotometric assay by determining the enzymatic activity of a captured analyte. The results showed that the method might be useful for label-free, fast, and sensitive detection of saccharides as well as glycoproteins over a wide concentration range.

Theoretical and experimental investigation of the focusing position in asymmetrical flow field-flow fractionation (AF4).

To better understand the focusing process and to determine the focusing position (zfoc) in Asymmetrical Flow Field-Flow Fractionation (AF4), theoretical and experimental studies were performed. A new theoretical expression that allows the calculation of zfoc in AF4 channels of any shape having an axial plane of symmetry was established. The equation was then applied to the particular case of a trapezoidal AF4 channel with tapered ends. The applicability of this equation was verified by comparing the calculated results with the experimental measures of the focusing position obtained using blue dextran dye under different flow conditions. The calculated values and the experimental results were in good agreement. The total uncertainty associated with the calculated zfoc was evaluated and was lower than 10%. A good repeatability and reproducibility of the focusing position was obtained. Moreover, the effects of the sample-injection position, the spacer thickness, the membrane aging, and the nature of the injected sample on the focusing position were investigated: zfoc was not influenced by these parameters, proving the universal applicability of the equation given in this work for predicting the focusing position, when the assumption of constancy of the cross-flow velocity at the membrane, which underlies this equation, is fulfilled. Additionally, it is notable that this theoretical expression is suitable for different models of AF4 channel and not peculiar to specific suppliers.

Silymarin prevents diabetes-induced hyperpermeability in human retinal endothelial cells / La silimarina previene la hiperpermeabilidad inducida por la diabetes en células endoteliales de retina humana

Introduction: Vascular endothelial growth factor (VEGF) plays an essential role in development of diabetic macular edema (DME). While there is evidence suggesting that silymarin, a flavonoid extracted from Silybum marianum, could be useful for prevention and treatment of diabetic nephropathy, no studies have been conducted in diabetic retinopathy (DR). The aim of this study was to assess the effect of silymarin on disruption of inner blood retinal barrier (BRB), the primary cause of DME. Materials and methods: Human retinal endothelial cells (HRECs) were cultured under standard (5.5mM D-glucose) and diabetogenic conditions (25mM D-glucose and 25mM D-glucose + recombinant vascular endothelial growth factor [rVEGF, 25mg/mL]). To assess cell viability, three concentrations of silymarin were tested (2, 4 and 10μg/mL). The effect of silymarin on HREC disruption was determined using a dextran (70kD) permeability asssay. Results: No differences were found in the viability of HRECs treated with 2 or 4μg/mL of silymarin as compared to untreated cells, but viability significantly decreased after using 10 μg/mL. The concentration of 4 μg/mL was therefore selected. Silymarin (4μg/mL) caused a significant decrease in VEGF-induced permeability in both media with 5.5nM (422±58 vs. 600±72 ng/mL/cm2; p<0.03) and 25nM of D-glucose (354 ± 28 vs. 567 ± 102 ng/mL/cm2; p<0.04). Discussion: Our results show that silymarin is effective for preventing hyperpermeability induced by diabetic conditions in HRECs. Further studies are needed to assess whether silymarin could be useful to treat DME (AU)

Introducción: El Vascular endothelial growth factor (VEGF) juega un papel esencial en el desarrollo del edema macular diabético (EMD). Existe evidencia que indica que el uso de la silimarina, extracto flavonoide del Silybum marianum, podría ser útil en la prevención y el tratamiento de la nefropatía diabética pero no se dispone de datos en retinopatía diabética (RD). El objetivo del estudio es evaluar el efecto de la silimarina sobre la disrupción de la barrera hematorretininana, que es la causa primaria del EMD. Material y métodos: Células endoteliales de retina humana (HRECs) se cultivaron en condiciones estándar (5.5mM de D-glucosa) y en condiciones suprafisiológicas de glucosa (25mM de D-glucosa y 25mM de D-glucosa + VEGF 25mg/dl). Para evaluar la viabilidad de las células se probaron 3 concentraciones de silimarina (2, 4 y 10μg/ml). El efecto de la silimarina sobre la disrupción de las HRECs se determinó mediante análisis de permeabilidad a dextrano (70kD). Resultados: No se observaron diferencias en la viabilidad de las HRECs tratadas con 2 o 4μg/ml de silimarina en comparación con las células no tratadas, pero se observó una reducción de la viabilidad con la concentración de 10μg/ml. Por consiguiente, se seleccionó la concentración de 4μg/ml de silimarina. La silimarina (4μg/ml) produjo un descenso significativo de la permeabilidad inducida por VEGF tanto en medio con 5.5mM de D-glucosa (422 ±58 vs. 600 ±72 ng/ml/cm2; p<0.03) como en medio con 25mM de D-glucosa (354±28 vs. 567±102 ng/ml/cm2; p<0.04). Discusión: Nuestros resultados demuestran que la silimarina es efectiva para prevenir la hiperpermeabilidad inducida por condiciones suprafisiológicas de glucosa en HRECs. Son necesarios más estudios para evaluar si la silimarina podría ser útil para el tratamiento del EMD (AU)

Non-invasive monitoring of in vivo hydrogel degradation and cartilage regeneration by multiparametric MR imaging.

Numerous biodegradable hydrogels for cartilage regeneration have been widely used in the field of tissue engineering. However, to non-invasively monitor hydrogel degradation and efficiently evaluate cartilage restoration in situ is still challenging. Methods: A ultrasmall superparamagnetic iron oxide (USPIO)-labeled cellulose nanocrystal (CNC)/silk fibroin (SF)-blended hydrogel system was developed to monitor hydrogel degradation during cartilage regeneration. The physicochemical characterization and biocompatibility of the hydrogel were evaluated in vitro. The in vivo hydrogel degradation and cartilage regeneration of different implants were assessed using multiparametric magnetic resonance imaging (MRI) and further confirmed by histological analysis in a rabbit cartilage defect model for 3 months. Results: USPIO-labeled hydrogels showed sufficient MR contrast enhancement and retained stability without loss of the relaxation rate. Neither the mechanical properties of the hydrogels nor the proliferation of bone-marrow mesenchymal stem cells (BMSCs) were affected by USPIO labeling in vitro. CNC/SF hydrogels with BMSCs degraded more quickly than the acellular hydrogels as reflected by the MR relaxation rate trends in vivo. The morphology of neocartilage was noninvasively visualized by the three-dimensional water-selective cartilage MRI scan sequence, and the cartilage repair was further demonstrated by macroscopic and histological observations. Conclusion: This USPIO-labeled CNC/SF hydrogel system provides a new perspective on image-guided tissue engineering for cartilage regeneration.

Anti-connective tissue growth factor detects and reduces plaque inflammation in early-stage carotid atherosclerotic lesions.

This study explored connective tissue growth factor (CTGF)-targeted ultrasmall superparamagnetic iron oxides (USPIOs) for noninvasive MRI of CTGF within carotid atherosclerotic lesions in apoE-deficient (apoE-/-) mice. Anti-CTGF polyclonal and nonspecific IgG antibodies were conjugated to polyethylene glycol-coated USPIOs, and apoE-/- carotid partial ligation-model mice were imaged via MRI before and after contrast administration. ApoE-/- mice were treated with CTGF-neutralizing antibodies for 3 weeks. Carotid artery diameter and plaque volume were measured via MRI in IgG and CTGF antibody-treated groups. Anti-CTGF-USPIO-treated macrophages showed the greatest iron uptake. MRI signal loss was observed in carotid atherosclerotic lesions 24 h after anti-CTGF-USPIO administration, consistent with the presence of nanoparticles, as indicated by pathological examinations. Atheromata in anti-CTGF-treated mice showed reduced macrophage deposition, CTGF expression, and plaque volume. Anti-CTGF-USPIOs can be used for the direct detection of CTGF and imaging of atherosclerotic lesions in vivo. CTGF is a potential therapeutic target for treating atherosclerosis.

Sensitive and non-invasive method for the in vivo analysis of membrane permeability in small animals.

Tissue membranes are boundaries that isolate organs or cavities in the body. These semi-permeable membranes are responsible for passive protection that acts through the regulation of nutrient absorption, secretion and filtration of small molecules. These functions could be altered as a consequence of inflammation or trauma, which in turn could lead to changes in permeability, allowing the entrance of toxins, antigens, proteins or facilitating the spread of tumors. Membrane permeability therefore plays an important role in numerous diseases. However, current experimental techniques that are available to quantify membrane permeability in small animals have limited precision and temporal specificity. Improvements in such measurements would lead to a deeper understanding of disease pathogenesis and this may accelerate the development of specific therapies. The study reported here concerns the efficacy of a novel, non-invasive imaging analysis-based measurement method that significantly improves the quantification of tissue membrane permeability in small animals, while at the same time mitigating the adverse effects experienced by the animals under study.

Functional food applications of dextran from Weissella cibaria RBA12 from pummelo (Citrus maxima).

Weissella cibaria RBA12 isolated from pummelo from Northeast India produces a dextran composed of 97% α-(1→6) linkages in the main chain and 3% α-(1→3) branched linkages. The in vitro prebiotic activity of dextran-RBA12 was explored. Dextran-RBA12 displayed enhanced growth of probiotic Bifidobacterium and Lactobacillus spp., and controlled growth of non-probiotic enteric bacteria. Dextran-RBA12 showed superior resistance to physiological barriers with a maximum hydrolysis of 0.51%, 0.31% and 0.24% by artificial gastric juice, α-amylase and intestinal fluid, respectively, whereas compared to maximum hydrolysis of 25.23%, 19.13% and 6%, respectively after 5h of incubation shown by commercial prebiotic inulin. The production of dextran from Weissella cibaria RBA12 in sourdough prepared from whole wheat flour, wheat bran and rye bran showed the highest dextran of 3.26±0.12% d.w. in rye bran. The overall study summarized that dextran-RBA12 can be used as a prebiotic and also can be easily produced in sourdough.

RBC aggregation in dextran solutions can be measured by flow cytometry.

The impact of macromolecules on RBC aggregation continues to be of interest, nevertheless present measurements still have limitations and need improvement. We applied flow cytometry to measure RBC aggregation in dextran T500 (Dx500) solution. The samples were fixed in the aggregated state by glutaraldehyde. Fixed RBC exhibit auto fluorescence, which can be detected by flow cytometry. Single cells, doublets, triplets and larger aggregates can be distinguished quantitatively and quickly due to the correlation between auto fluorescence intensity and number of RBC per measured event. With the increase in concentration of Dx500, percentages of all aggregates and bigger aggregates increased significantly at concentration of 2%, 4% and 6%, while decreased when the concentration reached 8% and 10%. The percentage of bigger aggregates in concentration of 4% was higher than that in 2% and 6%. The data of flow cytometry was confirmed by microscopic observation and are in good agreement with the literature. The method provide additional advantages to the conventional measurement of RBC aggregation. It gets the distribution of single cells and aggregates as derived from the microscopic observation with hematocrit of physiological level. It uses sample volume as 1/5∼1/10 as needed in sendimentation and photometricmethods.

Intestinal permeability induced by lipopolysaccharide and measured by lactulose, rhamnose and mannitol sugars in chickens.

Increased intestinal permeability (IP) can lead to compromised health. Limited in vivo IP research has been conducted in chickens. The objectives of the current study were to develop a model of increased IP utilizing lipopolysaccharide (LPS Escherichia coli O55:B5) and to evaluate IP changes using the lactulose, mannitol and rhamnose (LMR) sugar permeability test. In addition, fluorescein isothiocyanate dextran (FITC-d), d-lactate, zonula occludens (ZO-1) and diamine oxidase (DAO) permeability tests were employed. Male Ross chickens were reared until day 14 on the floor in an animal care facility and then transferred to individual cages in three separate experiments. In each of experiments 1 and 2, 36 chicks were randomly allocated to receive either saline (control) or LPS (n=18/group). Lactulose, mannitol and rhamnose sugar concentration in blood was measured at 0, 30, 60, 90, 120 and 180 min in experiment 1, at 60, 90 and 120 min in experiment 2 and at 90 min in experiment 3 (n=16/group). Lipopolysaccharide was injected intraperitoneally at doses of 0.5, 1 and 1 mg/kg BW in experiments 1, 2 and 3, respectively, on days 16, 18 and 20, whereas control received sterile saline. On day 21, only birds in experiments 1 and 2 were fasted for 19.5 h. Chicks were orally gavaged with the LMR sugars (0.25 gL, 0.05 gM, 0.05 gR/bird) followed by blood collection (from the brachial vein) as per time point for each experiment. Only in experiment 3, were birds given an additional oral gavage of FITC-d (2.2 mg/ml per bird) 60 min after the first gavage. Plasma d-lactate, ZO-1 and DAO concentrations were also determined by ELISA in experiment 3 (n=10). Administration of LPS did not affect IP as measured by the LMR sugar test compared with control. This was also confirmed by FITC-d and DAO levels in experiment 3 (P>0.05). The plasma levels of d-lactate were decreased (P<0.05). Plasma levels of ZO-1 were increased in the third experiment only and did not change in the first two experiments. Lipopolysaccharide at doses of 0.5 and 1 mg/kg did not increase IP in this model system. In conclusion, the LMR sugar can be detected in blood 90 min after the oral gavage. Further studies are needed for the applicability of LMR sugars tests.

Assessment of Dextran Antigenicity of Intravenous Iron Preparations with Enzyme-Linked Immunosorbent Assay (ELISA).

Intravenous iron preparations are typically classified as non-dextran-based or dextran/dextran-based complexes. The carbohydrate shell for each of these preparations is unique and is key in determining the various physicochemical properties, the metabolic pathway, and the immunogenicity of the iron-carbohydrate complex. As intravenous dextran can cause severe, antibody-mediated dextran-induced anaphylactic reactions (DIAR), the purpose of this study was to explore the potential of various intravenous iron preparations, non-dextran-based or dextran/dextran-based, to induce these reactions. An IgG-isotype mouse monoclonal anti-dextran antibody (5E7H3) and an enzyme-linked immunosorbent assay (ELISA) were developed to investigate the dextran antigenicity of low molecular weight iron dextran, ferumoxytol, iron isomaltoside 1000, ferric gluconate, iron sucrose and ferric carboxymaltose, as well as isomaltoside 1000, the isolated carbohydrate component of iron isomaltoside 1000. Low molecular weight iron dextran, as well as dextran-based ferumoxytol and iron isomaltoside 1000, reacted with 5E7H3, whereas ferric carboxymaltose, iron sucrose, sodium ferric gluconate, and isolated isomaltoside 1000 did not. Consistent results were obtained with reverse single radial immunodiffusion assay. The results strongly support the hypothesis that, while the carbohydrate alone (isomaltoside 1000) does not form immune complexes with anti-dextran antibodies, iron isomaltoside 1000 complex reacts with anti-dextran antibodies by forming multivalent immune complexes. Moreover, non-dextran based preparations, such as iron sucrose and ferric carboxymaltose, do not react with anti-dextran antibodies. This assay allows to assess the theoretical possibility of a substance to induce antibody-mediated DIARs. Nevertheless, as this is only one possible mechanism that may cause a hypersensitivity reaction, a broader set of assays will be required to get an understanding of the mechanisms that may lead to intravenous iron-induced hypersensitivity reactions.

High Efficiency Molecular Delivery with Sequential Low-Energy Sonoporation Bursts.

Microbubbles interact with ultrasound to induce transient microscopic pores in the cellular plasma membrane in a highly localized thermo-mechanical process called sonoporation. Theranostic applications of in vitro sonoporation include molecular delivery (e.g., transfection, drug loading and cell labeling), as well as molecular extraction for measuring intracellular biomarkers, such as proteins and mRNA. Prior research focusing mainly on the effects of acoustic forcing with polydisperse microbubbles has identified a "soft limit" of sonoporation efficiency at 50% when including dead and lysed cells. We show here that this limit can be exceeded with the judicious use of monodisperse microbubbles driven by a physiotherapy device (1.0 MHz, 2.0 W/cm(2), 10% duty cycle). We first examined the effects of microbubble size and found that small-diameter microbubbles (2 µm) deliver more instantaneous power than larger microbubbles (4 & 6 µm). However, owing to rapid fragmentation and a short half-life (0.7 s for 2 µm; 13.3 s for 6 µm), they also deliver less energy over the sonoporation time. This translates to a higher ratio of FITC-dextran (70 kDa) uptake to cell death/lysis (4:1 for 2 µm; 1:2 for 6 µm) in suspended HeLa cells after a single sonoporation. Sequential sonoporations (up to four) were consequently employed to increase molecular delivery. Peak uptake was found to be 66.1 ± 1.2% (n=3) after two sonoporations when properly accounting for cell lysis (7.0 ± 5.6%) and death (17.9 ± 2.0%), thus overcoming the previously reported soft limit. Substitution of TRITC-dextran (70 kDa) on the second sonoporation confirmed the effects were multiplicative. Overall, this study demonstrates the possibility of utilizing monodisperse small-diameter microbubbles as a means to achieve multiple low-energy sonoporation bursts for efficient in vitro cellular uptake and sequential molecular delivery.