Development of a coculture model of encapsulated cells.

In the whole animal, metabolic regulations are set by reciprocal interactions between various organs, via the blood circulation. At present, analyses of such interactions require numerous and uneasily controlled in vivo experiments. In a search for an alternative to in vivo experiments, our work aims at developing a coculture system in which different cell types are isolated in polymer capsules and grown in a common environment. The signals exchanged between cells from various origins are, thus, reproducing the in vivo intertissular communications. With this perspective, we evaluated a new encapsulation system as an artificial housing for liver cells on the one hand and adipocytes on the other hand. Murine hepatocytes were encapsulated with specially designed multicomponent capsules formed by polyelectrolyte complexation between sodium alginate, cellulose sulphate and poly(methylene-coguanidine) hydrochloride, of which the permeability has been characterized. We demonstrated the absence of cytotoxicity and the excellent biocompatibility of these capsules towards primary culture of murine hepatocytes. Encapsulated hepatocytes retain their specific functions--transaminase activity, urea synthesis, and protein secretion--during the first four days of culture in minimum medium. Mature adipocytes, isolated from mouse epidydimal fat, were embedded in alginate beads. Measurement of protein secretion shows an identical profile between free and embedded adipocytes. We finally assessed the properties of encapsulated hepatocytes, cryopreserved over a periods of up to four months. The perspective of using encapsulated cells in coculture are discussed, since this system may represent a promising tool for fundamental research, such as analyses of drug metabolism, intercellular regulations, and metabolic pathways, as well as for the establishment of a tissue bank for storage and supply of murine hepatocytes.

Isoelectric focusing of alpha-1 acid glycoprotein (orosomucoid) in immobilized pH-gradients with 8M urea: detection of its desialylated variants using an alkaline phosphatase-linked secondary antibody system.

A method allowing a clear separation of the different variants of desialylated alpha 1-acid glycoprotein (orosomucoid) has been developed using isoelectric focusing in immobilized pH gradients, supplemented with 8 M urea and 2% v/v 2-mercaptoethanol. Immunoblotting with two antibody-steps afforded high sensitivity and permitted the detection of about 700 pg of alpha 1-acid glycoprotein in a 20 microL plasma sample diluted 1:28 672. A one year old bloodstrain, kept at room temperature, could easily be phenotyped.

Urea nitrogen, creatinine, and uric acid levels in postmortem serum, vitreous humor, and pericardial fluid.

Urea nitrogen, creatinine, and uric acid are relatively stable in postmortem serum and may, therefore, be used for diagnostic purposes when chronic kidney disease and end-stage renal failure are investigated as causes of death. Nevertheless, uncertainties remain in defining the best alternative to postmortem serum for the identification and assessment of significantly decreased kidney function. In this study, we investigated urea nitrogen, creatinine, and uric acid levels in postmortem serum, pericardial fluid, and vitreous humor in a series of medico-legal cases (500 autopsies) with various causes of death. No postmortem interval-related differences were observed in any of the investigated fluids for any analyzed parameter, confirming the biochemical stability of all compounds after death. Data analysis failed to reveal statistically significant differences between postmortem serum and pericardial fluid urea nitrogen, creatinine, and uric acid concentrations. Conversely, statistically significant differences were observed in all analyzed biomarkers between postmortem serum and vitreous humor levels, with lower concentrations of all markers measured in vitreous. The results of this study suggest that, in order to estimate as accurately as possible blood analyte concentrations at the time of death, pericardial fluid should be preferred to vitreous humor.

Cross-sectional observational study of 208 patients with non-classical urea cycle disorders.

Urea cycle disorders (UCDs) are inherited disorders of ammonia detoxification often regarded as mainly of relevance to pediatricians. Based on an increasing number of case studies it has become obvious that a significant number of UCD patients are affected by their disease in a non-classical way: presenting outside the newborn period, following a mild course, presenting with unusual clinical features, or asymptomatic patients with only biochemical signs of a UCD. These patients are surviving into adolescence and adulthood, rendering this group of diseases clinically relevant to adult physicians as well as pediatricians. In preparation for an international workshop we collected data on all patients with non-classical UCDs treated by the participants in 20 European metabolic centres. Information was collected on a cohort of 208 patients 50% of which were ≥ 16 years old. The largest subgroup (121 patients) had X-linked ornithine transcarbamylase deficiency (OTCD) of whom 83 were female and 29% of these were asymptomatic. In index patients, there was a mean delay from first symptoms to diagnosis of 1.6 years. Cognitive impairment was present in 36% of all patients including female OTCD patients (in 31%) and those 41 patients identified presymptomatically following positive newborn screening (in 12%). In conclusion, UCD patients with non-classical clinical presentations require the interest and care of adult physicians and have a high risk of neurological complications. To improve the outcome of UCDs, a greater awareness by health professionals of the importance of hyperammonemia and UCDs, and ultimately avoidance of the still long delay to correctly diagnose the patients, is crucial.

Current concepts in the pathogenesis of urea cycle disorders.

The common feature of urea cycle diseases (UCD) is a defect in ammonium elimination in liver, leading to hyperammonemia. This excess of circulating ammonium eventually reaches the central nervous system, where the main toxic effects of ammonium occur. These are reversible or irreversible, depending on the age of onset as well as the duration and the level of ammonium exposure. The brain is much more susceptible to the deleterious effects of ammonium during development than in adulthood, and surviving UCD patients may develop cortical and basal ganglia hypodensities, cortical atrophy, white matter atrophy or hypomyelination and ventricular dilatation. While for a long time, the mechanisms leading to these irreversible effects of ammonium exposure on the brain remained poorly understood, these last few years have brought new data showing in particular that ammonium exposure alters several amino acid pathways and neurotransmitter systems, cerebral energy, nitric oxide synthesis, axonal and dendritic growth, signal transduction pathways, as well as K(+) and water channels. All these effects of ammonium on CNS may eventually lead to energy deficit, oxidative stress and cell death. Recent work also proposed neuroprotective strategies, such as the use of NMDA receptor antagonists, nitric oxide inhibitors, creatine and acetyl-l-carnitine, to counteract the toxic effects of ammonium. Better understanding the pathophysiology of ammonium toxicity to the brain under UCD will allow the development of new strategies for neuroprotection.

Inappropriate antidiuretic hormone secretion: long-term successful urea treatment.

Hyponatremia is the main complication of inappropriate antidiuretic hormone secretion (SIADH), sometimes fatal. Treatment strategy depends on the cause and the severity of the hyponatremia. Recent studies have shown the efficacy of urea in treating acute hyponatremia secondary to SIADH, by inducing an osmotic water drive. We describe an infant with chronic hyponatremia secondary to SIADH in which the long-term oral treatment with urea was successful and well tolerated. The aim of this paper is to highlight the potential benefits of urea treatment in case of chronic hyponatremia secondary to SIADH. CONCLUSION: Chronic oral urea treatment in children with SIADH allows an easy and safe water and sodium control and may permit a decrease in fluid restriction in this situation.

Orosomucoid (alpha-1 acid glycoprotein) phenotyping by use of immobilized pH gradients with 8 M urea and immunoblotting. A new variant encountered in a population study.

Orosomucoid (ORM) phenotyping has been performed on 329 unrelated Swiss subjects, using immobilized pH gradients with 8 M urea and 2% v/v 2-mercaptoethanol followed by immunoblotting. After desialylation the band patterns of ORM confirmed that the polymorphism of the structural locus ORM1 is controlled by three codominant autosomal alleles (ORM1*F1, ORM1*S and ORM1*F2). One rare and one new allele were detected. The rare variant, tentatively assigned to the second structural locus ORM2, is observed in a cathodal position and named ORM2 B1. The new variant, tentatively assigned to the first structural locus ORM1, is observed in a region located between ORM1 S and ORM1 F2, and named ORM1 F3. Moreover, the pI values of the ORM variants have been measured accurately with Immobiline Dry Plates (LKB): they were found to be within the pH range 4.93-5.14.

Morphogenesis of the ureterovesical junction:a histologic and microanatomic study in the rat.

OBJECTIVES: To investigate the development of the ureterovesical junction in rats. METHODS: A total of 110 albino rats (50 prenatal and 60 newborn) with a gestation of 21 days were studied at the age of 17 days after conception until 5 days after birth. The lower urinary tract was microdissected. Microphotography (110 animals), histologic examination (44 animals), and scanning electron microscopy (66 animals) of the ureterovesical junction were performed. Urea and creatinine from the amniotic fluid of 20 fetuses and from the urine of 10 neonates were measured. RESULTS: At day 17 after conception, separate penetration of the mesonephric duct and ureter into the wall of the urogenital sinus was observed. Continuity between the lumen of the ureter and the urogenital sinus was established on day 19 after conception. The straight passage of the intramural ureter into the urogenital sinus at day 17 after conception changed to the definitive L-shape with a vertical entry into the bladder on day 5 after birth. In the distal ureter, the change of the mesenchymal tissue into immature smooth muscle was first observed at birth, and the muscle became mature on the fifth postnatal day. At birth, Waldeyer's sheath was recognized. The creatinine and urea levels were stable prenatally (average 22.4 micromol/L and 6.88 mmol/L, respectively) and rose significantly postnatally (average 133 micromol/L and 32.65 mmol/L, respectively). CONCLUSIONS: The attachment of the ureter to the urogenital sinus and later to the bladder, the modification of its passage, and its mobility within Waldeyer's sheath may be essential in preventing vesicoureteral reflux. The production of urine and its flow does not seem to be the trigger of ureteral smooth muscle formation.

Promoter rearrangements cause species-specific hepatic regulation of the glyoxylate reductase/hydroxypyruvate reductase gene by the peroxisome proliferator-activated receptor alpha.

In liver, the glyoxylate cycle contributes to two metabolic functions, urea and glucose synthesis. One of the key enzymes in this pathway is glyoxylate reductase/hydroxypyruvate reductase (GRHPR) whose dysfunction in human causes primary hyperoxaluria type 2, a disease resulting in oxalate accumulation and formation of kidney stones. In this study, we provide evidence for a transcriptional regulation by the peroxisome proliferator-activated receptor alpha (PPARalpha) of the mouse GRHPR gene in liver. Mice fed with a PPARalpha ligand or in which PPARalpha activity is enhanced by fasting increase their GRHPR gene expression via a peroxisome proliferator response element located in the promoter region of the gene. Consistent with these observations, mice deficient in PPARalpha present higher plasma levels of oxalate in comparison with their wild type counterparts. As expected, the administration of a PPARalpha ligand (Wy-14,643) reduces the plasma oxalate levels. Surprisingly, this effect is also observed in null mice, suggesting a PPARalpha-independent action of the compound. Despite a high degree of similarity between the transcribed region of the human and mouse GRHPR gene, the human promoter has been dramatically reorganized, which has resulted in a loss of PPARalpha regulation. Overall, these data indicate a species-specific regulation by PPARalpha of GRHPR, a key gene of the glyoxylate cycle.

In vitro engineering of human stratified urothelium: analysis of its morphology and function.

PURPOSE: Gastric or intestinal patches, commonly used for reconstructive cystoplasty, may induce severe metabolic complications. The use of bladder tissues reconstructed in vitro could avoid these complications. We compared cellular differentiation and permeability characteristics of human native with in vitro cultured stratified urothelium. MATERIALS AND METHODS: Human stratified urothelium was induced in vitro. Morphology was studied with light and electron microscopy and expression of key cellular proteins was assessed using immunohistochemistry. Permeability coefficients were determined by measuring water, urea, ammonia and proton fluxes across the urothelium. RESULTS: As in native urothelium the stratified urothelial construct consisted of basal membrane and basal, intermediate and superficial cell layers. The apical membrane of superficial cells formed villi and glycocalices, and tight junctions and desmosomes were developed. Immunohistochemistry showed similarities and differences in the expression of cytokeratins, integrin and cellular adhesion proteins. In the cultured urothelium cytokeratin 20 and integrin subunits alpha6 and beta4 were absent, and symplekin was expressed diffusely in all layers. Uroplakins were clearly expressed in the superficial umbrella cells of the urothelial constructs, however, they were also present in intermediate and basal cells. Symplekin and uroplakins were expressed only in the superficial cells of native bladder tissue. The urothelial constructs showed excellent viability, and functionally their permeabilities for water, urea and ammonia were no different from those measured in native human urothelium. Proton permeability was even lower in the constructs compared to that of native urothelium. CONCLUSIONS: Although the in vitro cultured human stratified urothelium did not show complete terminal differentiation of its superficial cells, it retained the same barrier characteristics against the principal urine components. These results indicate that such in vitro cultured urothelium, after being grown on a compliant degradable support or in coculture with smooth muscle cells, is suitable for reconstructive cystoplasty.

Oxidative potential of a panel of carbonaceous and metallic nanoparticles

Characterisation of nanoparticles (NP) based on size distribution, surface area, reactivity, and aggregation status of nanoparticles (NP) are of prime importance because they are usually closely related to toxicity. To date, most of the toxicity studies are quite time and money consuming. In the present study we report the oxidative properties of a panel of various NP (four Carbonaceous, nine Metal oxides, and one Metal as showed in Table 1) assessed with an acellular reactivity test measuring dithiothreitol (DTT) consumption (Sauvain et al. 2008). Such a test allows determining the ability of NP to catalyse the transfer of electrons from DTT to oxygen. DTT is used as a reductant species. NP were diluted and sonicated in Tween 80® to a final concentration of 50 g/mL. Printex 90 was diluted 5 times before doing the DTT assay because of its expected higher activity. Suspensions were characterised for NP size distribution by Nanoparticle Tracking Analysis (Nanosight©). Fresh solutions were incubated with DTT (100 μM). Aliquots were taken every 5 min and the remaining DTT was determined by reacting it with DTNB. The reaction rate was determined for NP suspensions and blank in parallel. The mean Brownian size distribution of NP agglomerates in suspension is presented in Table 1. D values correspond to 10th, and 50th percentiles of the particle diameters. All the NP agglomerated in Tween 80 with a D50 size corresponding to at least twice their primary size, except for Al2O3 (300 nm). The DTT test showed Printex 90 sample to be the most reactive one, followed by Diesel EPA and Nanotubes. Most of the metallic NP was nonresponding toward this test, except for NiO and Ag which reacted positively and ZnO which presented the most negative reactivity (see Figure 1). This last observation suggests that electron transfer between DTT and oxygen is hindered in presence of ZnO compared with the blank. Such "stabilization" could be attributable to ZnO dissolution and complexation between Zn2+ ions and DTT.

On problems of calculating energy expenditure and substrate utilization from respiratory exchange data.

Indirect calorimetry based on respiratory exchange measurement has been successfully used from the beginning of the century to obtain an estimate of heat production (energy expenditure) in human subjects and animals. The errors inherent to this classical technique can stem from various sources: 1) model of calculation and assumptions, 2) calorimetric factors used, 3) technical factors and 4) human factors. The physiological and biochemical factors influencing the interpretation of calorimetric data include a change in the size of the bicarbonate and urea pools and the accumulation or loss (via breath, urine or sweat) of intermediary metabolites (gluconeogenesis, ketogenesis). More recently, respiratory gas exchange data have been used to estimate substrate utilization rates in various physiological and metabolic situations (fasting, post-prandial state, etc.). It should be recalled that indirect calorimetry provides an index of overall substrate disappearance rates. This is incorrectly assumed to be equivalent to substrate "oxidation" rates. Unfortunately, there is no adequate golden standard to validate whole body substrate "oxidation" rates, and this contrasts to the "validation" of heat production by indirect calorimetry, through use of direct calorimetry under strict thermal equilibrium conditions. Tracer techniques using stable (or radioactive) isotopes, represent an independent way of assessing substrate utilization rates. When carbohydrate metabolism is measured with both techniques, indirect calorimetry generally provides consistent glucose "oxidation" rates as compared to isotopic tracers, but only when certain metabolic processes (such as gluconeogenesis and lipogenesis) are minimal or / and when the respiratory quotients are not at the extreme of the physiological range. However, it is believed that the tracer techniques underestimate true glucose "oxidation" rates due to the failure to account for glycogenolysis in the tissue storing glucose, since this escapes the systemic circulation. A major advantage of isotopic techniques is that they are able to estimate (given certain assumptions) various metabolic processes (such as gluconeogenesis) in a noninvasive way. Furthermore when, in addition to the 3 macronutrients, a fourth substrate is administered (such as ethanol), isotopic quantification of substrate "oxidation" allows one to eliminate the inherent assumptions made by indirect calorimetry. In conclusion, isotopic tracers techniques and indirect calorimetry should be considered as complementary techniques, in particular since the tracer techniques require the measurement of carbon dioxide production obtained by indirect calorimetry. However, it should be kept in mind that the assessment of substrate oxidation by indirect calorimetry may involve large errors in particular over a short period of time. By indirect calorimetry, energy expenditure (heat production) is calculated with substantially less error than substrate oxidation rates.

Whole body protein metabolism and resting energy expenditure in pregnant Gambian women.

Whole body protein metabolism and resting energy expenditure (REE) were measured at 11, 23, and 33 wk of pregnancy in nine pregnant (not malnourished) Gambian women and in eight matched nonpregnant nonlactating (NPNL) matched controls. Rates of whole body nitrogen flux, protein synthesis, and protein breakdown were determined in the fed state from the level of isotope enrichment of urinary urea and ammonia during a period of 9 h after a single oral dose of [15N]glycine. At regular intervals, REE was measured by indirect calorimetry (hood system). Based on the arithmetic end-product average of values obtained with urea and ammonia, a significant increase in whole body protein synthesis was observed during the second trimester (5.8 +/- 0.4 g.kg-1.day-1) relative to values obtained both for the NPNL controls (4.5 +/- 0.3 g.kg-1.day-1) and those during the first trimester (4.7 +/- 0.3 g.kg-1.day-1). There was a significant rise in REE during the third trimester both in the preprandial and postprandial states. No correlation was found between REE after meal ingestion and the rate of whole body protein synthesis.

Comparison between computational alanine scanning and per-residue binding free energy decomposition for protein-protein association using MM-GBSA: application to the TCR-p-MHC complex.

Recognition by the T-cell receptor (TCR) of immunogenic peptides (p) presented by Class I major histocompatibility complexes (MHC) is the key event in the immune response against virus-infected cells or tumor cells. A study of the 2C TCR/SIYR/H-2K(b) system using a computational alanine scanning and a much faster binding free energy decomposition based on the Molecular Mechanics-Generalized Born Surface Area (MM-GBSA) method is presented. The results show that the TCR-p-MHC binding free energy decomposition using this approach and including entropic terms provides a detailed and reliable description of the interactions between the molecules at an atomistic level. Comparison of the decomposition results with experimentally determined activity differences for alanine mutants yields a correlation of 0.67 when the entropy is neglected and 0.72 when the entropy is taken into account. Similarly, comparison of experimental activities with variations in binding free energies determined by computational alanine scanning yields correlations of 0.72 and 0.74 when the entropy is neglected or taken into account, respectively. Some key interactions for the TCR-p-MHC binding are analyzed and some possible side chains replacements are proposed in the context of TCR protein engineering. In addition, a comparison of the two theoretical approaches for estimating the role of each side chain in the complexation is given, and a new ad hoc approach to decompose the vibrational entropy term into atomic contributions, the linear decomposition of the vibrational entropy (LDVE), is introduced. The latter allows the rapid calculation of the entropic contribution of interesting side chains to the binding. This new method is based on the idea that the most important contributions to the vibrational entropy of a molecule originate from residues that contribute most to the vibrational amplitude of the normal modes. The LDVE approach is shown to provide results very similar to those of the exact but highly computationally demanding method.