Revisiting definition and assessment of intestinal trans-epithelial passage.

This study aims to remind that Intestinal Passage (IP) measurement is a complex task that cannot be achieved by a unique measure of an orally given exogenous marker in blood or urine. This will be illustrated in the case of NOD mice. Indeed, various methods have been proposed to measure IP. Among them ex vivo measurement in Ussing chambers of luminal to serosal fluxes of exogenous markers and in vivo measurement of exogenous markers in blood or urine after oral gavage are the more commonly used. Even though they are commonly used indifferently, they do not give the same information and can provide contradictory results. Published data showed that diabetic status in female Non Obese Diabetic (NOD) mice increased FD4 concentration in blood after gavage but did not modify FD4 fluxes in Ussing chamber. We observed the same results in our experimental conditions and tracked FD4 concentrations in blood over a kinetic study (Area Under the Curve-AUC). In vivo measurements are a dynamic process and address not only absorption (IP and intestinal surface) but also distribution, metabolism and excretion (ADME). Diabetic status in NOD mice was associated with an increase of intestinal length (absorptive surface), itself positively correlated with AUC of FD4 in blood. We concluded that increased intestinal length induced by diabetic status will extend the absorptive surface and increase FD4 concentration in plasma (in vivo measurement) despite no modification on IP of FD4 (ex vivo measurement). In addition, this study characterized intestinal function in diabetic NOD mice. Diabetic status in NOD female mice increases intestinal length and decreases paracellular IP (FSS) without affecting transcellular IP (HRP, FD4). Histological studies of small and large intestine did not show any modification of intestinal circumference nor villi and crypt size. Finally, diabetic status was not associated with intestinal inflammation (ELISA).

A Multiparametric Assay Platform for Simultaneous In Vivo Assessment of Pronephric Morphology, Renal Function and Heart Rate in Larval Zebrafish.

Automated high-throughput workflows allow for chemical toxicity testing and drug discovery in zebrafish disease models. Due to its conserved structural and functional properties, the zebrafish pronephros offers a unique model to study renal development and disease at larger scale. Ideally, scoring of pronephric phenotypes includes morphological and functional assessments within the same larva. However, to efficiently upscale such assays, refinement of existing methods is required. Here, we describe the development of a multiparametric in vivo screening pipeline for parallel assessment of pronephric morphology, kidney function and heart rate within the same larva on a single imaging platform. To this end, we developed a novel 3D-printed orientation tool enabling multiple consistent orientations of larvae in agarose-filled microplates. Dorsal pronephros imaging was followed by assessing renal clearance and heart rates upon fluorescein isothiocyanate (FITC)-inulin microinjection using automated time-lapse imaging of laterally positioned larvae. The pipeline was benchmarked using a set of drugs known to induce developmental nephrotoxicity in humans and zebrafish. Drug-induced reductions in renal clearance and heart rate alterations were detected even in larvae exhibiting minor pronephric phenotypes. In conclusion, the developed workflow enables rapid and semi-automated in vivo assessment of multiple morphological and functional parameters.

Assessment of Histiotrophic Nutrition Using Fluorescent Probes.

Histiotrophic nutrition is a process whereby the rodent visceral yolk sac (VYS) internalizes exogenous macromolecules, degrades them, and sends the degradation products to the embryo. Quantification and visualization of histiotrophic nutrition can be accomplished using fluorescent tracer molecules such as fluorescein isothiocyanate-conjugated albumin (FITC-albumin). The methods are simple and can provide complimentary functional and structural information in studies of the effects of embryotoxicants on visceral yolk sac function.

Assessment of the Intestinal Barrier with Five Different Permeability Tests in Healthy C57BL/6J and BALB/cJ Mice.

BACKGROUND: Intestinal permeability is thought to be of major relevance for digestive and nutrition-related diseases, and therefore has been studied in numerous mouse models of disease. However, it is unclear which tools are the preferable ones, and how normal values should be defined. AIMS: To compare different in vivo permeability tests in healthy mice of commonly used genetic backgrounds. METHODS: We assessed the intestinal barrier in male and female C57BL/6J and BALB/cJ mice of different ages, using four orally administered permeability markers, FITC-dextran 4000 (FITC-D4000) and ovalbumin (OVA) measured in plasma, and polyethylene glycol (PEG) and lactulose/mannitol (Lac/Man) measured in urine, and by assessing lipopolysaccharide (LPS) in portal vein plasma. RESULTS: After gavage, FITC-D4000, OVA, Lac/Man, and PEG400, but not PEG4000, were detectable in plasma or urine. Female mice tended to have a higher permeability according to the FITC-D4000, OVA, and PEG400 tests, but the Lac/Man ratio was higher in males. No significant differences between the two mouse strains of young and old mice were observed except for mannitol recovery, which was higher in BALB/cJ mice compared to C57BL/6J mice (p < 0.05). Virtually no LPS was detected in healthy mice. For all markers, normal values have been defined based on 5th-95th percentile ranges of our data. CONCLUSION: Selected oral permeability tests, such as FITC-D4000, OVA, PEG400, and Lac/Man, as well as LPS measurements in portal vein plasma, could be suitable for the evaluation of the intestinal barrier in mice, if used in a standardized way.

A study of the transport and immobilisation mechanisms of human red blood cells in a paper-based blood typing device using confocal microscopy.

Recent research on the use of bioactive paper for human blood typing has led to the discovery of a new method for identifying the haemagglutination of red blood cells (RBCs). When a blood sample is introduced onto paper treated with the grouping antibodies, RBCs undergo haemagglutination with the corresponding grouping antibodies, forming agglutinated cell aggregates in the paper. A subsequent washing of the paper with saline buffer could not remove these aggregates from the paper; this phenomenon provides a new method for rapid, visual identification of the antibody-specific haemagglutination reactions and thus the determination of the blood type. This study aims to understand the mechanism of RBC immobilization inside the paper which follows haemagglutination reactions. Confocal microscopy is used to observe the morphology of the free and agglutinated RBCs that are labelled with FITC. Chromatographic elution patterns of both agglutinated and non-agglutinated RBCs are studied to gain insight into the transport behaviour of free RBCs and agglutinated aggregates. This work provides new information about RBC haemagglutination inside the fibre network of paper on a microscopic level, which is important for the future design of paper-based blood typing devices with high sensitivity and assaying speed.

Assessment of histiotrophic nutrition using fluorescent probes.

Histiotrophic nutrition is a process whereby the rodent visceral yolk sac (VYS) internalizes exogenous macromolecules, degrades them, and sends the degradation products to the embryo for use in de novo macromolecular biosynthesis. This process is important for embryonic development during early gestation prior to the formation of the functional placenta. Quantification and visualization of histiotrophic nutrition can be accomplished using fluorescent tracer molecules such as fluorescein isothiocyanate-conjugated albumin (FITC-albumin) that can be visualized using fluorescent microscopy and quantified using fluorescent spectroscopy. The methods are simple and can provide complementary functional and structural information in studies of the effects of embryotoxicants on yolk sac function.

In vitro assessment of alkylglyceryl-functionalized chitosan nanoparticles as permeating vectors for the blood-brain barrier.

A series of O-substituted alkylglyceryl chitosans with systematically varied alkyl chain length and degree of grafting has been employed for the formulation of aqueous nanoparticulate systems, which were in turn investigated for their effects on a modeled blood-brain-barrier system of mouse-brain endothelial cells. Barrier function measurements employing electric cell-substrate impedance sensing and analyses of tight junction-specific protein profiles have indicated that the alkylglyceryl-modified chitosan nanoparticles impact upon the integrity of the model blood-brain barrier, whereas confocal microscopy experiments have demonstrated the efficient cellular uptake and the perinuclear localization of these nanoparticles. The application of nanoparticles to the model blood-brain barrier effected an increase in its permeability, as demonstrated by following the transport of the tracer molecule fluorescein isothiocyanate.

Quantitative assessment of intestinal injury using a novel in vivo, near-infrared imaging technique.

Intestinal injury owing to inflammation, severe trauma, and burn is a leading cause of morbidity and mortality. Currently, animal models employed to study the intestinal response to injury and inflammation depend on outdated methods of analysis. Given that these classic intestinal assays are lethal to the experimental animal, there is no ability to study the gut response to injury in the same animal over time. We postulated that by developing an in vivo assay to image intestinal injury using fluorescent dye, it could complement other expensive, time-consuming, and semiquantitative classic means of detecting intestinal injury. We describe a novel in vivo, noninvasive method to image intestinal injury using a charge-coupled device (CCD) camera that allows for serial visual and quantitative analysis of intestinal injury. Our results correlate with traditional, time-consuming, semiquantitative assays of intestinal injury, now allowing the noninvasive, nonlethal assessment of injury over time.

A direct immunoassay assessment of streptavidin- and N-hydroxysuccinimide-modified biochips in validation of serological TNFalpha responses in hemophagocytic lymphohistiocytosis.

The authors report 2 biochip platforms on gold manufactured by either nanoscale biotinylated self-assembled architectures to streptavidin surface or proteins containing free NH(2) groups to N-hydroxysuccinimide (NHS)-activated surfaces and investigated the potential application of tumor necrosis factor-alpha (TNFalpha) serodiagnosis of hemophagocytic lymphohistiocytosis (HLH). Interactions of TNFalpha antigen and TNFalpha antibody on the biochips were optimized using an indirect immunofluorescence method. Variation coefficients were 1.87% to 4.56% on the streptavidin biochip and 5.03% to 8.64% on the NHS biochip. The correlation coefficients (r) in TNFalpha and TNFalpha antibody assays in HLH patients between the 2 biochip formats were 0.9623 and 0.9386 and the concordance frequencies were 92.2% and 96.1%, respectively. To detect plasma TNFalpha-receptor complexes (TNFR1 and R2) in HLH, a biochip assay strategy was developed. Plasma levels of TNFalpha, TNFalpha antibody, and TNFalpha-receptor complexes (TNFR1 and R2) were detected in plasmas from 42 HLH cases using streptavidin biochips. Frequencies of the biomarkers in the plasmas were 40.5% (17/42) for TNFalpha, 30.9% (13/42) for TNFalpha antibody, 28.6% (12/42) for TNFalpha-receptor 1 complex, and 26.1% (11/42) for TNFalpha-receptor 2 complex, respectively. The streptavidin biochip format was more sensitive than the NHS surface and was demonstrated to be a valuable tool to identify individual biomarker molecules and molecular complexes in sera and cell lysates and to track therapeutic progress of patients.

Assessment of lipopolysaccharide-binding activity of Bifidobacterium and its relationship with cell surface hydrophobicity, autoaggregation, and inhibition of interleukin-8 production.

This study was performed to screen probiotic bifidobacteria for their ability to bind and neutralize lipopolysaccharides (LPS) from Escherichia coli and to verify the relationship between LPS-binding ability, cell surface hydrophobicity (CSH), and inhibition of LPS-induced interleukin-8 (IL-8) secretion by HT-29 cells of the various bifidobacterial strains. Ninety bifidobacteria isolates from human feces were assessed for their ability to bind fluorescein isothiocyanate (FITC)-labeled LPS from E. coli. Isolates showing 30-60% binding were designated LPS-high binding (LPS-H) and those with less than 15% binding were designated LPS-low binding (LPS-L). The CSH, autoaggregation (AA), and inhibition of LPS-induced IL-8 release from HT-29 cells of the LPS-H and LPS-L groups were evaluated. Five bifidobacteria strains showed high levels of LPS binding, CSH, AA, and inhibition of IL-8 release. However, statistically significant correlations between LPS binding, CSH, AA, and reduction of IL-8 release were not found. Although we could isolate bifidobacteria with high LPS-binding ability, CSH, AA, and inhibition of IL-8 release, each characteristic should be considered as strain dependent. Bifidobacteria with high LPS binding and inhibition of IL-8 release may be good agents for preventing inflammation by neutralizing Gram-negative endotoxins and improving intestinal health.

Assessment of lectin-binding analysis for in situ detection of glycoconjugates in biofilm systems.

An assessment of lectin-binding analysis for the characterization of extracellular glycoconjugates as part of the extracellular polymeric substances in environmental microbial communities was performed using fully hydrated river biofilms. The applicability of the method was evaluated for single, dual and triple staining with a panel of fluor-conjugated lectins. It was shown that lectin-binding analysis was able to stain glycoconjugates within biofilm communities. Lectin staining also demonstrated spatial heterogeneity within the biofilm matrix. Furthermore, the application of two or even three lectins was possible if suitable combinations were selected. The lectin-binding analysis can be combined with general nucleic acid stains to collect both nucleic acid and glycoconjugate signals. The effects of incubation time, lectin concentration, fluor labelling, carbohydrate inhibition, order of addition and lectin interactions were studied. An incubation time of 20 min was found to be sufficient for completion of lectin binding. It was not possible to ascertain saturating concentration for individual lectins, therefore a standard concentration was used for the assay. Carbohydrate inhibition tests indicated that fluorescein isothiocyanate (FITC)-conjugated lectins had more specific binding characteristics than tetramethyl rhodamine isothiocyanate (TRITC)- or cyanine dye (CY5)-labelled lectins. The order of addition and the nature of the fluor conjugate were also found to influence the binding pattern of the lectins. Therefore the selection of a panel of lectins for investigating the EPS matrix must be based on a full evaluation of their behaviour in the biofilm system to be studied. Despite this necessity, lectin-binding analysis represents a valuable tool to examine the glycoconjugate distribution in fully hydrated biofilms. Thereby, chemical heterogeneities within extracellular biofilm locations can be identified in order to examine the role (e.g. sorption properties, microenvironments, cell-extracellular polymeric substance interactions) of the extracellular polymeric substances in environmental biofilm systems.

Effects of absorption enhancers on the transport of model compounds in Caco-2 cell monolayers: assessment by confocal laser scanning microscopy.

Three typical absorption enhancers, i.e., sodium caprate (Cap-Na), sodium deoxycholate (Deo-Na), and dipotassium glycyrrhizinate (Grz-K), were compared in terms of their permeability-enhancing effects on hydrophilic and hydrophobic model compounds in Caco-2 cell monolayers. The transepithelial electrical resistance (TEER) of the monolayers was reduced concentration-dependently by treatment with Cap-Na and Deo-Na, while treatment with Grz-K increased the TEER. Two patterns of TEER reduction were observed: one pattern indicated that Cap-Na had a rapid reducing effect, and another indicated that Deo-Na had a delayed reducing effect. These reductions in the TEER were accompanied by the increased transepithelial transport of two hydrophilic model compounds, sodium fluorescein (Flu-Na; MW = 376, log P = -1.52) and fluorescein isothiocyanate-dextran 4000 (FD-4; MW = 4400, log P = -2.0), and one hydrophobic model compound, rhodamine 123 hydrate (Rh123; MW = 381, log P = 1.13). The transport-enhancing effects of Cap-Na and Deo-Na on these model compounds decreased in the following order: FD-4 > Rh123 > Flu-Na, while Grz-K was found to have no effect on the transport of any of these model compounds. Confocal laser scanning microscopy (CLSM) of Caco-2 cell monolayers revealed that Cap-Na and Deo-Na enhanced the transepithelial transport of the hydrophilic model compounds via the paracellular route and that of the hydrophobic model compound via both paracellular and transcellular routes. Semiquantitative visual information obtained from CLSM images reflected the results of the transport experiment.

Analysis of protein binding to receptor-doped lipid monolayers by Monte Carlo simulation.

This paper presents a Monte Carlo simulation (MCS) method for estimating the parameters that characterize ligand-receptor binding directly from experimentally derived binding isotherms. Binding parameters are estimated by incorporating an MCS algorithm for ligand binding to a two-dimensional receptor array into a nonlinear regression program. The MCS method was tested by analyzing experimental isotherms of avidin binding to biotinylated lipid in Langmuir-Blodgett (LB) monolayers. The MCS-derived cooperativity coefficients and intrinsic association constants for avidin-biotin binding to LB films are correlated strongly (R2 > 0.93) with the binding parameters determined from the same experimental data by a thermodynamic equilibrium binding model (Zhao et al. 1993. Langmuir. 9:3166-3173). This result shows MCS to be an accurate and potentially more versatile method for characterizing biomolecular interactions at surfaces.