Morphologic and functional development of whole human fetal stomachs grafted into nude mice.

To study in vivo the cellular differentiation and secretion of human developing fetal stomach, ethically and technically impossible to perform in utero, 256 fetal stomachs were xenografted. Human stomachs from 6- to 10-week-old fetuses were grafted for 1-273 days into nude mice. Biopsies for immunohistochemistry, hybridization and electron microscopy were taken and a catheter introduced into the human stomach. Macroscopic growth was fast and cells in S phase were numerous during the first 9 weeks, then the stomach size was stable and the gastric mucosa, of adult type, remained normal. In situ hybridization detected only a minute mouse mesenchymal chimerism in the graft. Chromogranin A, intrinsic factor and H+/K+ adenosine triphosphatase were immunohistolocally detected in epithelial cells 20 days after grafting, gastrin was detected after 30 days and pepsinogen after 60 days. The pH in gastric juice, which was at 8.0 +/- 0.1 from days 10-25, dropped from 4.39 +/- 1.80 at 30 days to 1.58 +/- 0.29 at 90 days. Intrinsic factor was stable and pepsin ranged from 6.8 +/- 7.8 to 134 +/- 51 units at 90 days. The differentiation of the epithelial cells in xenografts was very accelerated in comparison to that in utero.

Acid secretion and response to pentagastrin or omeprazole in human fetal stomach xenografts.

BACKGROUND: The dual capacity of stomach tissue to secrete acid and to respond to secretagogues is indicative of the terminal stages of gastric functional maturation. In this study 6- to 10-week-old human fetal stomachs xenografted into nude mice were used to study parietal cells' functional maturation. METHODS: Thirty-four transplants were microsurgically grafted either inside a pouch created on the nude peritoneum (n = 15) or on the host stomach and esophagus (n = 19). The mucosa of transplanted tissues was analyzed by immunohistochemical techniques to detect gastric cells. Gastric cell secretions were collected before and after pentagastrin or omeprazole treatment. RESULTS: Parietal, G, and D cells were detected immunohistochemically only after 1 month of grafting. All xenografts actively secreted acid after 1 or 2 months' transplantation at each graft site. Acid secretion was significantly stimulated by intraperitoneally injected pentagastrin (mean pH +/- SD, 3.2 +/- 0.7 vs. 2.0 +/- 0.5; n = 10, P = 0.005) and was dramatically inhibited by intragastrically administered omeprazole (2.3 +/- 0.6 vs. 6.5 +/- 0.7; n = 15, P = 0.0007) after 5 hours. CONCLUSION: Stomach xenografts were able to develop normally. Parietal cells were physiologically mature with functional proton pumps and active gastrin receptors, as demonstrated after omeprazole and pentagastrin treatment, respectively. Because stomach xenografts matured very rapidly, it is possible that a stomach xenograft model can be used for further studies on the functional maturation of human gastric epithelial cells, as well as the factors that influence this maturation in humans.

Gastric penetration of amoxicillin in a human Helicobacter pylori-infected xenograft model.

The delivery of antibiotics into Helicobacter pylori-infected human stomachs is still poorly understood. Human embryonic gastric xenografts in nude mice have recently been proposed as a new model for the study of H. pylori infection. Using this model, we compared the penetration of amoxicillin, after intraperitoneal administration of a dose of 20 mg/kg of body weight, into the gastric mucosae of infected and uninfected xenografts. The concentrations of this drug in serum and superficial gastric mucosae were determined at 20 min and 1 and 3 h after injection. Ten mice with H. pylori-infected grafts (n = 5) or uninfected grafts (n = 5) were studied. Mucosal samples were obtained by cryomicrotomy. The concentrations in serum were similar to those obtained in the serum of humans after oral administration of 1 g of amoxicillin. The mean area under the tissue concentration-versus-time curve from 0 to 3 h obtained for mice with infected grafts was significantly higher than that obtained for the animals with uninfected grafts (P = 0.01). These results suggest that the penetration of amoxicillin into the superficial gastric mucosa may be substantially increased in the case of H. pylori infection. Thus, human xenografts in nude mice represent a new, well-standardized model for investigation of systemic delivery of drugs into H. pylori-infected gastric mucosa.

Assimilation of [57Co]-labeled cobalamin in human fetal gastrointestinal xenografts into nude mice.

Cobalamin (Cbl) and its Cbl-binding proteins are present in amniotic fluid. Because amniotic fluid is swallowed by the embryo-fetus, we studied the ability of Cbl to be transported and metabolized across the embryo-fetal digestive tract. Human embryonic stomachs and intestines were transplanted into nude mice. The basal secretion of Cbl-binding proteins was studied by gel filtration of the graft juices. Intrinsic factor (IF) was looked for in gastric mucosa by immunohistochemistry. The uptake of [57Co]-labeled Cbl by the intestinal graft was studied by Schilling tests and HPLC. IF, haptocorrin, and a transcobalamin-like protein were detected in gastric juice, with concentration ranges of 5.0-26.4, 1.9-27.1, and 5.2-12.6 pmol/mL, respectively. The IF [57Co]Cbl complex had a single isoprotein with a pI at 5.6, which was maintained after incubation with neuraminidase. Urine excretion percentages (Schilling tests) ranged from 5.5 to 21.2% and from 0.3 to 1.6% when cyano-[57Co]Cbl-IF or cyano-[57Co]Cbl, respectively, was instilled in intestinal grafts. Chloroquine reduced significantly the percentage of excreted [57Co]Cbl. The [57Co]Cbl was mainly excreted as cyano-[57Co]Cbl in urines, showing a low coenzyme conversion. In conclusion, IF is secreted by the nonstimulated embryonic stomach and lacks sialic acid. Cbl binds to it and is subsequently transported across the xenografted embryo-fetal intestine. This suggests that amniotic fluid may contribute to Cbl delivery to the embryo-fetus.

Human embryonic gastric xenografts in nude mice: a new model of Helicobacter pylori infection.

In vitro or animal models have been used to investigate the pathogenesis of Helicobacter pylori infection. However, extrapolation to humans of results obtained with these heterologous models remains difficult. We have developed a new model for the study of H. pylori infection that uses human entire embryonic stomachs engrafted in nude mice. At 80 days after implantation, 22 of these xenografts, which exhibited a mature gastric epithelium, were inoculated with 10(7) to 10(8) CFU of either H. pylori LB1, a freshly isolated H. pylori strain (n = 12), or H. pylori ATCC 49503 (n = 10). After 12-week examination, H. pylori LB1 persistently colonized the antrum of all inoculated grafts, as assessed by culture (mucus and mucosa), immunohistochemistry (mucosa), and a rapid urease test (mucus). H. pylori ATCC 49503, either before or after in vivo passage, permitted only a transient 2-week colonization in one of the five inoculated grafts in both groups. Colonization was always associated with an increase of gastric juice pH. A mild neutrophil infiltration of the gastric mucosa was noted solely in infected grafts. Transmission electron microscopy showed adherence of H. pylori organisms to epithelial cell surface. In six animals, intracytoplasmic location of this bacterium was observed in the antrum or the fundus. These results allow us to propose this model as a new ex vivo model for the study of specific H. pylori-gastric cell interactions.

[Amplification of immunologic reactions using catalytic deposition at the reaction sites of tyramine derivatives. A decisive gain in sensitivity in immunohistochemistry and in situ hybridization]. / L'amplification des réactions immunologiques par dépôt catalytique aux sites de réaction de dérivés de la tyramine. Un gain décisif de sensibilité en immunohistochimie et en hybridation in situ.

In the catalyzed reporter deposition technique, horseradish peroxidase catalyzes the activation of conjugated phenolic compounds resulting in the covalent binding of the radicalized intermediates to electron rich moieties in the protein molecules present at the site of reaction. Comparing the biotinyl-, digoxigeninyl-, and fluoresceinyl-derivatives of tyramine and four immunohistochemical formats, we showed that the most sensitive detection system was that using biotinyl-tyramide and an immunohistochemical technique using a biotinylated secondary antibody followed by a streptavidin peroxidase. Amplification without background staining was obtained in most biotin rich tissues with digoxigeninyl-tyramide. With fluoresceinyl-tyramide, clear signal amplification was observed in the fluorescent microscope. Finally, when compared with standard methods, increased sensitivity was obtained with the fluorescent derivative for detection of hybridized sequences in interphase chromosomes.

Gastric diffusion of antibiotics used against Helicobacter pylori.

Only a few pharmacological studies have been carried out on men and guinea pigs to determine the gastric diffusion of antibiotics, which are active against Helicobacter pylori. The results of these studies have been analysed in considering the physicochemical nature, the mode of administration, the way of gastric diffusion (topic and/or systemic) and the pharmacological interactions. The correlation of these pharmacokinetic results with those obtained in clinical trials is difficult because of the heterogeneity of the pharmacological and pharmacodynamic data. The absence of a convenient and suitable animal or in vitro study model renders further standardized pharmacokinetic studies in infected man and at steady state necessary.