Lysozyme and mucins in gastric adenomas.

A method for the simultaneous demonstration of lysozyme and mucins in 39 cases of gastric adenomas differentiated two intermediate cell types. The first was similar to a columnar cell comprising a single cell population which covered extensive areas of the adenomas. This cell type often showed supranuclear lysozyme reactivity and apical neutral mucins, sialomucins, and sulphomucins in variable amounts. The second cell type was found in 11 adenomas, located mainly in the fundal area. It seemed to be a transitional form between the goblet cell and the Paneth cell. This cell type was scattered among columnar cells, occasional Paneth-like cells, and small goblet cells. These two types of intermediate cells may be regarded as abnormally differentiated integral elements of gastric adenomas. They may be associated with the neck stem cells in the cytogenesis of gastric adenomas.     

In vitro binding of Helicobacter pylori to human gastric mucin.

The in vitro binding of four Helicobacter pylori strains to human gastric mucin was studied with an enzyme-linked immunosorbent assay. All four strains were found to bind to purified mucin. Neuraminidase treatment and nonspecific oxidation of mucin decreased bacterial adherence to the macromolecule. Mucin preparations were also found to inhibit attachment of H. pylori to HEp-2 monolayers.     

In vitro glucose consumption in severe hyperleukocytosis. A cause of factitious hypoglycemia

Following the observation, in a patient with leukemia, of a factitious hypoglycemia that was related to in vitro glucose consumption by leukocytes, a study of glucose fall in vitro in the blood of 17 patients with leukemia of various types and with various grades of leukocytosis was undertaken. Results show that in vitro glucose consumption is linked to the intensity of leukocytes concentration, and is independent of the type of leukocyte. Factitious hypoglycemia may thus frequently occur in patients with high blood leukocyte count. This phenomenon, that was previously described, is again of interest to day because many laboratories do not yet use any more glycolysis inhibitors that partially inhibit it. Indeed, these inhibitors interfere with measures of other biological parameters that are simultaneously determined in autoanalysers.     

In vitro primate gastric mucosa: electrical characteristics

Ion transport by the resting, isolated, rhesus gastric mucosa was assessed under conditions of minimal diffusion limitation to oxygen by 1) the substitution of Na+ and Cl- of the bathing solutions with less permeant ions, 2) the drugs amiloride and ouabain, and 3) estimation of net fluxes of 22Na by methods designed to circumvent the problem of poorly matched tissues. The mucosae developed potential differences of 51.3 +/ 3.5 mV, serosal side positive and had conductances of 5.56 +/- 0.30 mS x cm-2. The permeabilities of the tissues to D-mannitol were between 7.80 x 10(-7) and 3.15 x 10(-7) cm x s-1. The relatively high conductance of this epithelium in the absence of significant edge damage and a low (32%) paracellular conductance stems mainly from a passive permeability to Cl-; active absorption of Na+ and active secretion of Cl- contribute equally to the short-circuit current. The mucosal entry step for Na+ is amiloride sensitive, whereas the serosal exit step can be inhibited by ouabain. The entry step for Cl- at the serosal membrane is possibly sodium dependent.     

In vitro surface properties of the newly recognized gastric pathogen Helicobacter pylori.

There appears to be a particular association between Helicobacter pylori and the gastric antrum, but the mechanisms by which the organism adheres to and colonizes the gastric mucosa are unclear. Surface hydrophobicity and surface charge mediate the adherence of other bacterial pathogens to mucosal epithelial cell surfaces. Therefore, in this study we characterized both the surface hydrophobicity and the surface charge of 10 H. pylori strains grown in broth culture. Four complementary methods were used to determine hydrophobicity: hydrophobic interaction chromatography, the salt aggregation test, comparison of bacterial adherence to polystyrene with adherence to sulfonated polystyrene, and measurement of contact angle with droplets of water. Three of the methods (salt aggregation test, adherence to polystyrene, and contact angles) indicated that each of the 10 strains expressed a relatively hydrophilic cell surface. In contrast, hydrophobic interaction chromatography determinations with both phenyl- and octyl-Sepharose suggested that the H. pylori strains were relatively hydrophobic. However, tetramethyl urea (0.4 M) did not reduce the binding of H. pylori to phenyl-Sepharose columns. DEAE-cellulose ion-exchange chromatography showed that each of the 10 strains of H. pylori had a surface which, overall, was highly negatively charged. We conclude that H. pylori expresses an overall relatively hydrophilic and negatively charged surface in vitro.     

In vitro effects of drugs on production of mucins in rabbit tracheal epithelial cells expressing mucin gene

Rabbit tracheal epithelial cells, cultured on collagen-coated dishes in serumfree and hormone-supplemented medium, were found to incorporate [³H]glucosamine into high-molecular-weight components that were secreted in the medium. The chemical analysis of the secreted products resulted in a profile that resembled that of mucous glycoproteins (mucins). When examined by dot blot analysis, the total RNA isolated from these cells hybridized to an antisense 30-mer oligonucleotide corresponding to a rat intestine mucin peptide sequence, indicating that mucin gene was expressed in these cell lines. Lung and liver tissues of rabbit did not express this gene. Transmission electron microscopy exhibited secretory granules in these cells. The incorporation of [³H]glucosamine into mucins was inhibited by three aryl-N-acetyl-galactosaminides and a chemical carcinogen,N-nitroso-N-ethyl urea, whereas 5-azacytidine enhanced the proliferation of cells as well as the radiolabeling of mucins. Parasympathetic agent (pilocarpine), cholinergic antagonist (atropine), and-adrenergic agonist (isoproterenol) alone have little effect on the secretion of mucins. The cholinergic agonist, methacholine, was found to increase the production of mucins and addition of atropine to the medium before methacholine blocked this stimulation. Histamine was found to stimulate mucin production in these cells.The opinions or assertions contained herein are the private views of the authors and are not to be construed as official or as reflecting the views of the Department of the Army or the Department of Defense.     

In vivo and in vitro lipidation of recombinant immunogens for direct iscom incorporation

We have previously reported strategies for Escherichia coli production of recombinant immunogens fused to hydrophobic tags to improve their capacity to be incorporated into an adjuvant formulation (J. Immunol. Methods 222 (1999) 171; 238 (2000) 181). Here, we have explored the possibility to use in vivo or in vitro lipidation of recombinant immunogens as means to achieve iscom incorporation through hydrophobic interaction. For the in vivo lipidation strategy, a general expression vector was constructed encoding a composite tag consisting of a sequence (lpp) of the major lipoprotein of E. coli, fused to a dual affinity fusion tag to allow efficient recovery by affinity chromatography. Upon expression in E. coli, fatty acids would be linked to the produced gene products. To achieve in vitro lipidation, the target immunogen would be expressed in frame with an N-terminal His6-ABP affinity tag, in which the hexahistidyl tag was utilized to obtain lipidation via a Cu2+-chelating lipid. A 238 amino acid segment DeltaSAG1, from the central region of the major surface antigen SAG1 of Toxoplasma gondii, served as model immunogen in this study. The two generated fusion proteins, lpp-His6-ABP-DeltaSAG1 and His6-ABP-DeltaSAG1, both expressed at high levels (approximately 5 and 100 mg/l, respectively), could be recovered to high purity by ABP-mediated affinity chromatography, and were evaluated in iscom-incorporation experiments. The His6-ABP-DeltaSAG1 fusion protein was associated to iscom matrix with pre-incorporated chelating lipid. Both fusion proteins were found in the iscom fractions after analytical ultracentrifugation in a sucrose gradient, indicating successful iscom incorporation/association. Iscom formation was further supported by electron microscopy analysis. In addition, these iscom preparations were demonstrated to induce high-titer antigen-specific antibody responses upon immunization of mice. For this particular target immunogen, DeltaSAG1, the induced antibodies demonstrated poor reactivity to the native antigen, although slightly better for the preparation employing the in vitro lipidation strategy, indicating that DeltaSAG1 was suboptimally folded or presented. Nevertheless, we believe that the presented strategies offer convenient alternative ways to achieve efficient adjuvant incorporation for recombinant immunogens.     

Variants of gastric carcinomas according to immunohistochemical mucins and CD10 expressions

Mucins and glycoprotein CD10 expression in 55 carcinomas of the stomach has been studied by immunohistochemistry. According to a profile of mucins and CD10 expression, 3 immunophenotypic variants of carcinoma were founded: gastric, intestinal and gastro-intestinal. Gastric and gastro-intestinal variants were more frequently than intestinal one. The correlation between the expression of investigated markers and histological type of carcinoma wasn't revealed The estimation of Ki-67 index has shown that the highest proliferative activity (>20%) in carcinomas with gastric phenotype has been found more often than in gastro-intestinal ones.     

Effects of oral glucose intake on gastric myoelectrical activity and gastric emptying.

To investigate the effect of oral glucose intake on gastric motility, we measured gastric myoelectrical activity and gastric emptying on two test conditions: 1) glucose intake and 2) water intake in the same 10 healthy male volunteers (20 to 29 years old). Gastric motility was evaluated with cutaneous-recorded electrogastrography (EGG) for 30 min both on fasting and after glucose or water intake, while gastric emptying was measured using acetaminophen-absorption method. There were no significant changes in EGG dominant frequency after water intake, but the frequency increased significantly after glucose intake. A postprandial dip (i.e., a transient decrease in frequency immediately after the food intake) was observed in 3 subjects after water intake and in 8 subjects following glucose intake. The EGG power ratio was significantly larger after glucose than water intake, with delayed gastric emptying in the former case. These results suggest that glucose is one of the components responsible for postprandial gastric motility.     

In vitro thymidine uptake and incorporation into thymic and bursal lymphocytes from young hypothyroidic chickens

Experiments were conducted to evaluate the ability of thymic and bursal lymphocytes from hypothyroidic chicks to take up 3H-thymidine. To produce hypothyroidism, chicks were fed a diet containing thiouracil (0.1%) from day of hatch to 3 weeks of age. At 1, 2 and 3 weeks of age, body weights were significantly lower for hypothyroid chicks, and at 2 and 3 weeks of age, the relative weights of the thymus and bursa were significantly less than those of controls. Furthermore, chicks treated with thiouracil had significantly lower serum concentrations of 3,5,3'-triiodothyronine (T3) and thyroxine (T4). Thymic cells from hypothyroid chicks incorporated less 3H-thymidine at 1 and 3 weeks of age as assessed by liquid scintillation counting and autoradiography. Liquid scintillation counts for labeled bursal cells from hypothyroid chicks were not lower at 1 week of age, but the counts were lower at 3 weeks of age. Autoradiographic studies with bursal cells revealed that 3H-thymidine incorporation was less, but not significantly less, in medium-size lymphocytes from thiouracil-treated chicks at both 1 and 3 weeks of age. Thymidine incorporation into large bursal cells was affected more by thiouracil treatment at 1 than 3 weeks of age.     

Biosynthesis of Entamoeba histolytica proteophosphoglycan in vitro

A complex glycoconjugate proteophosphoglycan (PPG) is present on the surface of the pathogenic protozoan parasite Entamoeba histolytica but not in the non-pathogenic Entamoeba dispar. It is thought to be an important molecule involved in pathogenesis. In order to study its biosynthesis, an in vitro cell-free system was developed. The specificity of the system was demonstrated by various criteria including immunoprecipitation by a specific monoclonal antibody. The in vitro synthesized molecule was found to be susceptible to mild acid hydrolysis, digestion by phosphoinositol-specific phospholipase C and nitrous acid deamination, the salient features for a PPG-like molecule. The in vitro product was not synthesized when heat-treated cellular-extract was used in the assay or when the cell extract was prepared from Entamoeba invadens, a species that lacks these glycoconjugates. Analysis of the glycan side chains of the in vitro synthesized product by thin layer chromatography revealed side chains of variable sizes including a fraction greater than six glycan units. The crude membranes used in the cell-free system were further fractionated by sucrose density gradient centrifugation. The fraction containing the PPG synthesizing activity when used in the assay resulted in a 10-fold increase in specific activity. Development of this cell-free system will facilitate further studies on the nature of intracellular organelles and the pathways that are involved in PPG biosynthesis.     

Oxygenation of frog gastric mucosa in vitro.

We have recently shown that 5% CO2/95% O2 in the serosal bathing solution, with 100% O2 in the mucosal solution, results in CO2-diffusion limitation of acid secretion in bullfrog gastric mucosa. Changing to 10% CO2/90% 02 on both surfaces doubles the acid secretory rate. We calculate that, were the rate of oxygen consumption to increase significantly as a result of secretory stimulation, the tissue would now be oxygen limited. This prediction is tested by raising the P02 by increasing the total pressure in a hyperbaric chamber. Since no change in acid secretory rate or potential difference was observed upon changing from PO2 = 0.9 to PO2 = 1.9 atm, we conclude that the tissue is not O2 limited at normal pressure. Decreasing PO2 below 0.9 atm, by contrast, decreases the acid secretory rate and raises both PD and resistance. We infer that the rate of oxygen consumption did not rise significantly when acid secretion was increased by supplying sufficient CO2.     

Isotopic labeling of mouse interferon by incorporation of radioactive amino acids during synthesis

Mouse interferon produced by C-243 cells induced with Newcastle disease virus was isotopically labeled by adding either [³⁵S]methionine or a ¹⁴C-labeled amino acid mixture to the culture medium. A method combining butyric acid and theophylline treatment and resulting in high interferon yields was used. Following purification by two-step affinity chromatography on poly(U) and antibody columns, the resulting material was analyzed on SDS-PAGE. The migration pattern of radioactivity and interferon coincided well and autoradiography revealed three major bands at migration distances corresponding, respectively, to 35, 28, and 22 K. Interferon represented 3.8% of all [³⁵S]methionine-labeled proteins and 2.6% of all ¹⁴C-amino acid-labeled proteins released into the medium.     

In vitro and in vivo effects of ammonia on glucose metabolism in the astrocytes of rat cerebral cortex.

Effects of 1 and 5 mM ammonium acetate on glucose metabolism were studied in astrocytes. But for an elevation in the levels of fructose-6-phosphate, phosphoenol pyruvate, and pyruvate, glucose metabolism was unaltered in the presence of 1 mM ammonium acetate. With 5 mM ammonium acetate, but for unaltered lactate, ADP, ATP and decreased aspartate, levels of several intermediates were elevated. Similar results were obtained when astrocytes isolated from hyperammonemic rats were incubated with glucose except for an enhanced production of 14CO2 from [U-14C]glucose. It is suggested that glucose metabolism of astrocytes may not be severely affected in astrocytes of cerebral cortex in acute hyperammonemic states.     

In vitro induction of delayed-type hypersensitivity.

Murine spleen cells, cultured in vitro for 6 days in the presence of high concentrations of burro erythrocytes (BRBC), are sensitized to exhibit delayed-type hypersensitivity (DTH) specific for this antigen. Such cells, on being injected with antigen into the footpads of normal mice, cause a 24-h swelling reaction. This activity of the cultured cells requires the presence of BRBC both during the in vitro incubation and in the footpad. The activity of the sensitized cells in causing swelling is sensitive to anti-Thy-1 antibody and complement, and the kinetics of the swelling reaction are characteristic of a DTH response. In vivo low-dose priming of the spleen cell donors considerably enhances the ability of the cultured cells to cause swelling. This system provides a means of studying the regulation of the induction of DTH in vitro.     

Measurement of antigen specific lymphocyte proliferation using 5-bromo-deoxyuridine incorporation An easy and low cost alternative to radioactive thymidine incorporation

The classical in vitro assay for the determination of cell mediated immune responses is the lymphocyte transformation test (LTT) in which cell proliferation is measured by incorporation of radioactive labeled thymidine (³H-TdR). The LTT assay using ³H-TdR is less suited for modestly equipped laboratories as it is costly, laborious and involves the need to handle radioactive isotopes and specialized equipment.

Here we describe an improved alternative LTT method which is capable of detecting specific cellular immune reactions (CMI) against (mycobacterial) antigens in vitro. This assay, the bromodeoxyuridine-ELISA LTT test, is simple, less expensive, reproducible and is as sensitive as the ³H-TdR test. The specific advantages of the test are a simple denaturation step and the fact that no radioactive isotopes are needed. The test is specifically suited for research laboratories in tropical countries which study CMI in those human infectious diseases where this arm of the immune response plays a pivotal role in the generation of immunity, e.g., in tubercolosis, leprosy and leishmaniasis.