Polyclonal antibodies for immunochemical determination of human pepsinogens.

The human gastric mucosa contains two main groups of aspartic proteinases, pepsin (EC 3.4.23.1) and gastricsin (EC 3.4.23.3), which differ by their structural, kinetic and immunological characteristics. The ratios between human aspartic proteases are important from the diagnostic point of view. Rabbit polyclonal antisera against human pepsinogen A and pepsinogen C (progastricsin) were obtained and tested for clinical purposes. Immunoblotting procedure seems to be a simple and sufficiently sensitive method for qualitative determination of pepsinogens in human gastric mucosa.

Changes in the zymogenic cell populations of the abomasa of sheep infected with Haemonchus contortus.

The effects of dietary urea supplementation and of a 10-week trickle infection regime, simulating chronic exposure to Haemonchus contortus, on the zymogenic population of the abomasa of Hampshire Down lambs was examined. At necropsy a variety of parameters including plasma pepsinogen concentration, the wet weights of abomasal fundic mucosal pieces and the amounts of pepsinogen contained in them, were assessed. Tissue pepsinogen concentration was measured as the total, acid-stable proteolytic activity present in mucosal homogenates, as well as immunohistochemically. The immunohistochemical findings were quantified using computer-aided image analysis. Elevation of plasma pepsinogen concentrations in infected animals was of borderline significance (P = 0.06). The fundic mucosae of infected animals were heavier (P < 0.02) than those of control animals, but there was no overall change in the pepsinogen content of tissues. Immunohistochemistry revealed that infected animals had increased numbers of zymogenic cells, due to mucous cell hyperplasia and the adaptation of cells to produce both mucins and pepsinogen. The pepsinogen content of chief cells, the major source of pepsinogen in uninfected animals, was reduced in infected lambs. Image analysis confirmed that at a mid-point of the mucosa of infected animals there was increased pepsinogen-specific immunoreactivity that corresponded with areas of mucosal hyperplasia. Mucous cell hyperplasia might therefore allow the maintenance of pepsinogen secretion in infected animals even if chief cell output is reduced.

Immunocytochemistry and in situ hybridization studies of pepsinogen C-producing cells in developing rat fundic glands.

The ontogeny of pepsinogen C-producing cells in rat fundic glands was studied by means of light and electron microscopy using an antiserum raised against a synthetic peptide based on rat pepsinogen C. To confirm the immunocytochemistry results, the expression of rat pepsinogen C messenger RNA (mRNA) in the fundic gland was also examined by in situ hybridization using a digoxigenin-labeled RNA probe. In adult rats, pepsinogen C was produced by chief cells, mucous neck cells, and intermediate mucopeptic cells. Pepsinogen C-producing cells appeared in embryos as early as 18.5 days' gestation. The development of these cells could be classified into four stages: (1) 18.5 days' gestation to 0.5 days after birth; (2) 0.5 days to 2 weeks after birth; (3) 3-4 weeks after birth; (4) 4-8 weeks after birth. In embryos and young animals, pepsinogen C-producing cells were mucopeptic cells. By 4 weeks after birth, mucous neck cells could be distinguished morphologically. The maturation stages of the chief cells could be traced by electron microscopy along the longitudinal axis of the rat fundic gland by double-staining with anti-pepsinogen C antibody and periodic acid-thiocarbohydrazide-silver proteinate. Positive reactions for pepsinogen C and pepsinogen C mRNA expression were detected in mucous neck cells. Therefore, we conclude that mucous neck cells are precursor cells of chief cells. Mucous neck cells, intermediate cells, and chief cells are in the same differentiating cell lineage.

Immunofluorometric assay of pepsinogen C and preliminary clinical applications.

We developed mouse monoclonal antibodies (Abs) against pepsinogen C with highly purified antigen isolated from gastric mucosa. The Abs were used to construct a two-site sandwich-type assay for pepsinogen C with time-resolved fluorometry as a detection technique. The assay has a detection limit of 0.1 microgram/L and is precise (within-run and day-to-day CVs < 11%). We used this assay to measure pepsinogen C in seminal plasma, breast cyst fluid, amniotic fluid, male and female serum, serum from patients with prostate cancer, urine, breast tumor cytosolic extracts, breast milk, and cerebrospinal fluid. Highest pepsinogen C concentrations were in seminal plasma, followed by breast cyst fluid and amniotic fluid. We found no correlation between prostate-specific antigen concentrations and concentrations of pepsinogen C in serum of prostate cancer patients, and concluded that this marker is not useful for either diagnosing or monitoring prostatic carcinoma. The availability of a highly sensitive, reliable, and convenient method for quantifying pepsinogen C will allow investigations into the possible diagnostic value of this analyte in various clinical conditions, including benign breast diseases, breast cancer, fertility, and pregnancy.

Ostrich pepsinogens I and II: purification, activation and chemical and immunochemical characterization of the enzymes from the proventriculus.

Pepsins are a series of gastric proteases secreted as inactive precursors (pepsinogens) which are active at acidic pH. The aim of this study was to purify ostrich pepsin(ogen)s and to compare their biochemical and immunological characteristics with those of pepsin(ogen)s of mammalian and avian origin. Ostrich pepsinogens were purified by ammonium sulphate fractionation, Toyopearl Super Q-650S chromatography and rechromatography, and hydroxylapatite chromatography of a pH 8.0 mucosal extract. Pepsins were obtained through acidification, and purified by chromatography on SP-Sephadex C-50. Amino acid compositions, N-terminal sequences, Ouchterlony double-diffusion as well as Western blot analysis were performed. Two pepsinogens were isolated and purified from the proventriculus of the ostrich, pepsinogens I and II. Both pepsinogens and pepsins were purified to homogeneity as shown by PAGE and SDS-PAGE, with SDS-PAGE revealing M(r) values of 40,400 and 41,900 for pepsinogens I and II, respectively. SDS-PAGE revealed M(r) values of 36,000 and 36,300 for ostrich pepsins I and II, respectively. Ostrich pepsinogens I and II were found to have identical N-terminal sequences, with Asp as N-terminal amino acid. Amino acid compositions were obtained for both pepsinogens, with ostrich pepsinogen I being slightly smaller in size with a total of 356 residues compared to 371 for ostrich pepsinogen II. Pepsinogen II showed a pI of 4.29. Ostrich pepsinogens I and II were found to be immunologically separate entities, and no cross-reactivity was observed between anti-(ostrich pepsinogen I/II) sera and porcine pepsin/pepsinogen. The study indicates that only two pepsinogens are present in the ostrich. They differ in terms of electrophoretic mobility, molecular mass and immunological reactivity, but have been found to have identical N-terminal sequences. It is concluded that both pepsinogens belong to the pepsinogen A class of aspartyl proteases (EC 3.4.23.1).

A new method for bovine pepsinogen purification. Preparation of a specific antibody.

Bovine pepsinogen was purified from abomasum by ammonium sulphate precipitation and ionic exchange chromatography on DEAE-cellulose and Mono Q columns. Purified pepsinogen was shown to be homogeneous by analytical electrophoresis, having an estimated molecular mass of 46,000 Daltons. The isoelectric point, determined by analytical chromatofocusing was 4.6. Using this pepsinogen preparation to immunize rabbits, a specific antiserum of high titer was obtained.

Occurrence of autoantibodies against intrinsic factor, H,K-ATPase, and pepsinogen in atrophic gastritis and rheumatoid arthritis.

The occurrence of autoantibodies against intrinsic factor, H,K-ATPase, and pepsinogen was analysed by means of enzyme-linked immunosorbent assay in three groups of sera. Group 1 comprised sera from 14 rheumatoid arthritis patients with normal acid secretion; group 2, sera from 18 rheumatoid arthritis patients with reduced acid secretion; and group 3, sera from 11 patients with pernicious anaemia or achylia. Groups 1 and 2 were rheumatoid factor-positive, and group 3 was negative. Intrinsic factor autoantibodies were low in groups 1 and 2. In group 3, 9 of the 11 sera (82%) scored positive. The highest titres of H,K-ATPase and pepsinogen autoantibodies were found in groups 2 and 3. Only one serum in group 1 scored positive against H,K-ATPase, and two against pepsinogen, whereas corresponding values were 11 (61%) and 7 (39%) in group 2, and 10 (91%) and 6 (55%) in group 3. Autoantibodies against H,K-ATPase from a pool of patient sera recognized both the alpha- and beta-subunits of the enzyme. The present results support the hypothesis of an autoimmune disease overlap between non-organ-specific rheumatoid arthritis and organ-specific pernicious anaemia.

Ductal cancers of the pancreas frequently express markers of gastrointestinal epithelial cells.

It has been found by immunohistochemical staining that antigens normally found in gastric and/or intestinal epithelial cells are expressed in most differentiated duct cell carcinomas of the pancreas. Among 88 such tumors, 93% and 92%, respectively, expressed M1 and cathepsin E, markers of gastric surface-foveolar epithelial cells, 51% expressed pepsinogen II, a marker of gastroduodenal mucopeptic cells, 48% expressed CAR-5, a marker of colorectal epithelial cells, and 35% expressed M3SI, a marker of small intestinal goblet cells. Most of the tumors also expressed normal pancreatic duct antigens; 97% expressed DU-PAN-2, and 59% expressed N-terminus gastrin-releasing peptide. In agreement with these findings, electron microscopy revealed malignant cells with fine structural features of gastric foveolar cells, gastric mucopeptic cells, intestinal goblet cells, intestinal columnar cells, pancreatic duct epithelial cells, and cells with features of more than one cell type. Normal pancreatic duct epithelium did not express any marker of gastrointestinal epithelial cells, whereas such benign lesions as mucinous cell hypertrophy and papillary hyperplasia commonly expressed gut-type antigens but rarely expressed pancreatic duct cell markers. By contrast, lesions characterized by atypical papillary hyperplasia commonly expressed both gastric and pancreatic duct cell markers. Metaplastic pyloric-type glands expressed pepsinogen II and, except for their expression of cathepsin E, were indistinguishable from normal pyloric glands. In marked contrast, the immunohistochemical and ultrastructural features of 14 ductuloacinar cell tumors were those of cells lining terminal ductules, centroacinar cells, and/or acinar cells; none expressed any gut-type antigen. The results indicate that gastrointestinal differentiation is common in both benign and malignant lesions of pancreatic duct epithelium and suggest that duct cell carcinomas are histogenetically related to gastric- and intestinal-type metaplastic changes of epithelial cells lining the main and interlobular ducts of the pancreas.

The occurrence of auto-antibodies in patients with gastro-duodenal lesions.

The occurrence of auto-antibodies in patients with the autoimmune disease pernicious anaemia and in patients with active duodenal ulcers was investigated. In order to characterize antigenic structures, various cellular and subcellular fractions were prepared from pig gastric mucosa and from a homogenate of duodenal mucosa. By means of an enzyme-linked immunosorbent assay and immunoblotting, both the H+,K(+)-ATPase and pepsinogen/pepsin were shown to constitute the major antigens. All of the seven pernicious-anaemia sera that were tested contained auto-antibodies against both antigens, and the epitopes of the H+,K(+)-ATPase were shown to be localized on its cytoplasmic face. In 75% (18/24) of the sera from patients with duodenal ulcers, auto-antibodies were detected when using purified antigens. Six sera reacted with H+,K(+)-ATPase and twelve reacted with pepsinogen, one reacted with both antigens, and four sera reacted with the duodenal mucosal antigen. The occurrence of auto-antibodies indicates that there is a mucosal lesion and that immunological factors may be involved in the pathogenesis of the disease in some patients.

The occurrence of gastric and duodenal auto-antibodies in peptic ulcer disease.

The possible relationship between peptic ulcer and the occurrence of auto-antibodies was investigated by means of an enzyme-linked immunosorbent assay (ELISA). Sera from 24 patients with active duodenal ulcer were analysed using cells and subcellular fractions from pig gastric acid duodenal mucosa for binding of immunoglobulins. Four sera (17%) reacted with a homogenate from duodenal mucosa. Nine sera (38%) were found to contain auto-antibodies against gastric mucosal cells. The cell-reactive auto-antibodies were shown to bind preferentially to parietal cells and chief cells. In these cells the antigens were identified as H, K-ATPase and pepsinogen respectively. Six sera were positive against purified H,K-ATPase; 12 sera were positive against pepsinogen, and only one of these sera reacted with both H,K-ATPase and pepsinogen. The results show that auto-antibodies are formed in a large fraction of patients (18/24; 75%) with peptic ulcer disease. The present study further demonstrates that enrichment of antigenic structures is required for obtaining a satisfactory sensitivity in the assay.

Characterization of antigenic structures in auto-immune atrophic gastritis with pernicious anaemia. The parietal cell H,K-ATPase and the chief cell pepsinogen are the two major antigens.

Using isolated cells and subcellular fractions from pig gastric mucosa, antigenic structures with specific binding of IgG from sera of patients with auto-immune atrophic gastritis were characterized by means of immunoblotting and enzyme-linked immunosorbent assay. In immunoblotting experiments using mucosal cells as the antigen source, two dominating bands of 94 and 41 kDa were found. The two major antigens were identified as the H,K-ATPase (94 kDa), which constitutes the parietal cell acid pump, and pepsinogen (41 kDa) located in the chief cells. There was also a small but significant binding of antibodies to a preparation of Na,K-ATPase, an enzyme which is about 60% homologous to H,K-ATPase. Commercial preparations of hog gastric pepsinogen and pepsin bound pernicious anaemia IgG with equal efficacy. When sera from seven patients with the diagnosis pernicious anaemia were tested, all were found to contain auto-antibodies against H,K-ATPase as well as pepsinogen. In intact, isolated H,K-ATPase-containing vesicles the cytosolic part of the ATPase molecule is facing the outside of the vesicles. Both intact and trypsinized vesicles were incubated with patient sera and with a monoclonal antibody against H,K-ATPase. Pernicious anaemia IgG was found to bind to a cytosolic, trypsin-resistant structure, but the binding of the monoclonal antibody was lost upon trypsinization. The present results indicate that intracellular structures of the gastric mucosa, due to cell damage, may be exposed to immune-competent cells, which do not recognize these structures as 'self'.

Seminal pepsinogen C is not identical with, but is very similar to gastric pepsinogen C.

Human seminal pepsinogen C has been purified and compared with gastric pepsinogen C. The two zymogens cannot be distinguished by amino acid compositions and sequences of the first 28 N-terminal amino acid residues are identical. Apparent immunological identity is observed with polyclonal antisera. Monoclonal antibodies toward seminal pepsinogen C have been produced. One is able to recognize a non-carbohydrate antigenic determinant only present in seminal pepsinogen C.

A new polymorphic pepsinogen locus (Pg-2) in the rat (Rattus norvegicus).

Only two types of pepsinogens, which are products of the Pg-1 locus, are present in rat urine. In gastric mucosa, however, additional pepsinogen isozymes are expressed. We have found a polymorphism for rat gastric mucosa pepsinogen using agarose gel electrophoresis. Some inbred rat strains expressed a pepsinogen band, while others did not. The trait was found to be controlled by a single autosomal locus. We tentatively designated the locus as Pg-2 with two alleles, Pg-2a for the one controlling presence of the band and Pg-2o for the one controlling absence. Linkage analysis using BN and TM strains revealed that Pg-2 was closely linked to Pg-1 (3.7 +/- 1.8 cM), and that it did not belong to LG I (Hbb and p), LG II (Acon-1 and Mup-1), LG IV (Hao-1 and Svp-1), LG V (Es-1 and Es-3), LG VI (Gc and h), LG IX (RT1), LG X (Fh and Pep-3), nor a LG containing Ahd-2 (as yet undetermined).

Pepsinogens: an update of biochemical, physiological, and clinical aspects.

In this paper the role of pepsinogen has been reviewed in its physiological and clinical aspects. Although acid secretion has traditionally received far more attention clinically and has therefore been studied in great detail, the development of cellular systems has recently seen a revival in interest of pepsinogen secretion. These systems have made it possible to study pepsinogen secretion in more detail. Although many questions remain unanswered, a picture of a stimulus-secretion coupling mechanism of the chief cell has emerged that resembles in many aspects the pancreatic acinar cell, but also possesses some unique features of its own. The chief cell monolayer culture has also made it possible to study pepsinogen synthesis, and these studies seem to have solved the old controversy of whether or not modulation of pepsinogen synthesis occurs as a result of increased secretion. It now seems that pepsinogen synthesis does indeed increase in response to stimulated secretion. In addition to physiological studies, this review has discussed clinical aspects of the human pepsinogens in various gastric disorders. The clinical implications of genetic heterogeneity of the human pepsinogens are especially intriguing. Relationships between certain PGA phenotypes and certain gastric disorders have been described and some studies have tried to evaluate the relevance of these findings for diagnostic purposes. So far, it seems that PGA phenotyping alone has only limited diagnostic value, but, in combination with serum PGA determinations, could be of additional help in the diagnosis of gastric malignancy. In addition, various studies suggest that the ratio of serum PGA and PGC levels may be helpful in determining the histological status of the gastric mucosa. A very promising possibility in solving the many problems involved in exact genotype determinations through phenotyping is the recent availability of cDNA probes. With this technique, the question of whether the association between PGA phenotypes and gastric malignancy is primary or secondary may be solved in the near future. In view of the very poor prognosis for gastric cancer, further studies concerning the relationships between gastric cancer, serum pepsinogen levels, and PGA phenotypes or genotypes will hopefully lead to the possibility of an earlier diagnosis for gastric malignancy.

Enzyme-linked immunosorbent assay of serum pepsinogen I.

A hybridoma monoclonal antibody against human pepsinogen I was used to develop an enzyme-linked immunosorbent assay for pepsinogen I in serum. In the two-step competitive procedure using antimouse immunoglobulin F(ab')2 fragment coupled to alkaline phosphatase, the measurable assay range was 8-256 micrograms/l. No cross-reactivity with rat pepsinogen 1, human pepsinogen II, gastrin I, bombesin, somatostatin and peptide YY was shown. However, there was slight cross-reactivity (0.09%) with porcine pepsinogen. The coefficients of variation within and between series were 7.6% and 13.0%. This enzyme-linked immunosorbent assay for serum pepsinogen I correlated positively with radioimmunoassay (r = 0.87, n = 92). The concentration range of serum pepsinogen I in 354 healthy controls was 15-100 micrograms/l with a lognormal distribution. Serum pepsinogen I levels were significantly higher in the subjects who developed active duodenal ulcer or active gastric ulcer, but significantly lower in those who had gastric cancer, than in control subjects.

Pepsinogen-like immunoreactivity among vertebrates: occurrence of common antigenicity to an anti-chicken pepsinogen antiserum in stomach gland cells of vertebrates.

Stomachs of 14 species selected from five classes of vertebrate were surveyed concerning the reactivity to an anti-adult chicken pepsinogen antiserum (anti-ACPg) with indirect immunofluorescence method. Gland cells of all these stomachs showed reactivity to the antiserum. Crude extract of stomachs from five representatives of mammals, birds, amphibians and fish showed peptic activity (at pH 2.2) of which 70-90% were pepstatin-sensitive. Zymogram and immunoblotting of crude extract revealed that the anti-ACPg-reactive proteins have peptic activity. Molecular weights of anti-ACPg-reactive proteins determined by immunoblotting coincided with the values of purified pepsinogens previously reported for these animals. These results indicate that pepsinogens have been conserved well during vertebrate evolution.

Immunochemical, electrophoretic, and genetic heterogeneity of pepsinogen I. Characterization with monoclonal antibodies.

Two immunologic subclasses of human pepsinogen I alpha-PG I and beta-PG I, have been identified based on their reactivity toward a murine monoclonal antibody that recognizes an epitope on the alpha-PG I isozymogens. The antibody was used to purify the major alpha- and beta-isozymogens from gastric mucosa and to determine their contributions to the previously described genetic polymorphism of PG I. The alpha-epitope was localized to the pepsin region of the molecules. The two major alpha-PG I isozymogens (Pg 3 alpha and Pg 5 alpha) and the major beta-PG I isozymogen (Pg 4 beta) were demonstrated to contain net charge differences located in the respective pepsin and activation peptide regions. We propose that the alpha- and beta-subclasses contain net charge amino acid substitutions encoded by the corresponding pepsinogen genes: PGA3, PGA4, and PGA5.

Immunohistochemical demonstration of induction of pyloric glands with low pepsinogen 1 (Pg 1) content in rat stomach by N-methyl-N'-nitro-N-nitrosoguanidine.

Three groups of male Fischer rats were given single doses of N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) at 160 mg (group 1), 80 mg (group 2) and 40 mg (group 3)/kg body weight by gastric intubation. A fourth group was given drinking water containing 100 micrograms/ml of MNNG for 2 weeks, and a fifth group served as a control. Rats were killed in weeks 5, 8 and 12. Serial sections of the pyloric mucosa were examined by paradoxical concanavalin A (Con A) staining and pepsinogen isozyme 1 (Pg 1) immunostaining. All pyloric glands contained class III mucin as detected by paradoxical Con A staining. Most pyloric glands had a high Pg 1 content, but a few stained only weakly if at all. The percentage and number (No./500 normal-looking pyloric glands) of pyloric glands with a low Pg 1 content were 50.0 and 0.2 +/- 0.4 (week 5), 87.5 and 0.5 +/- 0.4 (week 8) and 100.0 and 1.2 +/- 1.0 (week 12) in group 1, 50.0 and 0.2 +/- 0.3 (week 8) and 87.5 and 0.5 +/- 0.4 (week 12) in group 2, and 30.0 and 0.2 +/- 0.4 (week 12) in group 4. No pyloric glands with a low Pg 1 content were found in groups 3 and 5. Thus the results showed significant dose-dependent induction (P less than 0.05-0.01) of altered pyloric glands demonstrating reduced Pg 1 content and their earlier appearance in groups given higher doses of MNNG. The results suggest that the appearance of pyloric glands with a low Pg 1 content may be a preneoplastic change in gastric carcinogenesis.

[Immunohistologic study of the localization of organ-specific antigens of the stomach and intestine and carcinoembryonic antigen in normal and pathologically altered stomach tissues]. / Immunogistologicheskoe izuchenie lokalizatsii organospetsificheskikh antigenov zheludka i kishechnika i rakovo-émbrional'nogo antigena v normal'nykh i patologicheski izmenennykh tkaniakh zheludka.

Localization of organospecific gastric and intestinal antigens, as well as of carcinoembryonic antigen (CEA) was studied by an indirect immune peroxidase method on the sections of fetal stomach, normal definitive stomach, gastric mucosa with features of superficial and deep gastritis, enterolysis and dysplasia, as well as in gastric tumours. Normally, pepsinogen was found to localize in zymogen cells and to disappear in enterolysis and dysplasia of gastric mucosa. Intestinal antigen is absent from the normal mucosa, but is found in all the cases with enterolysis and dysplasia. CEA is most specific for dysplasia of gastric epithelium. In cancers of intestinal type pepsinogen was found in 54%, intestinal (colonic) antigen in 37.5%, CEA in 62.5%. In diffuse type cancers pepsinogen was absent, intestinal antigen was found in 76.9%, CEA in 92% of tumours.