Pepsinogen identification in the middle ear fluid of children with otitis media with effusion.

OBJECTIVES: This study aims to investigate the presence and concentration of pepsin/pepsinogen in middle ear fluid and to discuss the potential mechanisms involved in the pathogenesis of this condition. PATIENTS AND METHODS: A total of 33 children (21 boys, 12 girls; mean age 5.7±2.4 years; range 3 to 13 years) diagnosed with otitis media with effusion and scheduled for operation were enrolled into the study. Fluids aspirated from the middle ear were assessed for the presence of pepsinogen and albumin and blood samples were drawn simultaneously for comparison. RESULTS: Mean pepsinogen concentration was statistically significantly higher in middle ear fluids compared with serum samples (262.4 ng/mL [range: 211.7 ng/mL - 301.1 ng/mL] versus 102.6 ng/mL [range: 80.7 ng/mL - 134.5 ng/mL], respectively) (p<0.001). On the other hand, mean albumin concentration was significantly lower (1.1 g/dL [range: 0.01 g/dL - 9.5 g/dL] versus 5.8 g/dL [range: 0.9 - 9.5 g/dL], respectively) (p<0.001). The highest pepsinogen concentration was detected in patients with purulent effusion (275.3 ng/mL). CONCLUSION: Our findings support the theory of gastro-esophageal reflux related pepsinogen transition to the middle ear and indicate that pepsinogen may a reliable biochemical marker for the assessment of gastro-esophageal reflux.

Detection of gastric pepsin in middle ear fluid of children with otitis media.

OBJECTIVE: We sought to confirm the finding of pepsin/pepsinogen in the middle ear fluid of children with otitis media in a larger sample size using a sensitive and specific pepsin assay. STUDY DESIGN AND SETTING: We evaluated 152 children (225 ear samples) in a prospective study at a tertiary care children's hospital. The presence of pepsin in middle ear aspirates was determined using enzymatic assay. RESULTS: Of the patients, 14.4 percent (22 of 152) had detectable pepsin activity in one or both of the ear samples with no pepsin activity detected in control serum. Average pepsin concentration in the samples was 96.6 +/- 170.8 ng/ml, ranging from 13 to 687 ng/ml. Pepsin concentration in the middle ear of children younger than 1.0 year was significantly higher than in older age groups. CONCLUSION AND SIGNIFICANCE: Results indicate that pepsin/pepsinogen is present in the middle ears of children with otitis media, although not at the high rate previously reported. Gastric reflux may be one causative factor in the pathogenesis of otitis media.

Pepsin assay: a marker for reflux in pediatric glue ear.

OBJECTIVE: To evaluate if analysis of pepsin/pepsinogen in middle ear effusions can be considered a diagnostic marker for laryngopharyngeal reflux (LPR) in children with otitis media with effusion (OME). MATERIAL AND METHODS: Ambulatory 24-hour dual-probe pH monitoring was carried out on 31 children with OME. Middle ear effusions were collected from 17 children during myringotomy. Total pepsin/pepsinogen concentrations in effusions were measured by ELISA using antipepsin antibody. RESULTS: Dual-probe pH monitoring showed that 22/31 (71%) of the studied children had significant LPR. The concentrations of pepsin/pepsinogen in middle ear effusions, ranged from 0.085 to 5.02 microg/ml, were found to be up to 4.5 to 231.44 times higher than the serum levels. There was a significant positive correlation between the level of pepsin/pepsinogen assayed in the effusions of the 17 children and the number of pharyngeal reflux episodes measured by pH monitoring. CONCLUSIONS: Control of LPR may be an essential component in the successful management of OME in pediatric patients. Pepsin/pepsinogen analysis in effusions of children, using ELISA, can be considered a reliable marker for assessment of reflux in children with OME.

[The functional status of the human stomach in the course of the experiment with antiorthostatic hypokinesia of 4 months duration].

The paper deals with type of changes in gastroduodenal organs associated with hypersecretion of the stomach induced by simulation of some effects of microgravity. Subjects in the investigation were 13 males volunteered for a 4-month bedrest study. Biochemical changes in blood, urine and gastric juices were analyzed in comparison with ultrasonic images of the gastroduodenum organs and data of stomach and duodenum endoscopy. The investigations showed increased blood and urine levels of pepsinogen indicative of gastric hypersecretion characterized by more intensive secretion of gastric juices, lowered pH, elevated pyloric tone, activation of duodenal secretion and cholepoiesis. Gastric hypersecretion was concomittant to the development of plethora in the abdominal veins. The highest gastric secretion was observed after two months of bed rest. Further bed rest extension was marked by fading of the signs of hypersecretion by the stomach and appearance of some signs of chronic stress. Our results expand the knowledge of changes in the gastroduodenal organs during bed rest, space flights and other conditions provocative of the hypersecretory transformation of the stomach.

"Familial hyperpepsinogenemia" and Helicobacter pylori infection.

OBJECTIVE: Pepsinogen 1 (PG1) is a proenzyme precursor to pepsin, a protease secreted by the gastric chief cell. PG1 levels correlate with maximal gastric acid output. In 1979, Rotter et al. reported two pedigrees in which elevated PG1 levels and duodenal ulcers were prevalent. They proposed autosomal dominant inheritance of elevated PG1 and suggested that it was a risk factor for duodenal ulcer disease. In 1982, Helicobacter pylori (Hp) was discovered and was shown to be an important factor in peptic ulcer disease. Hp infection is also associated with increased PGI levels. We tested serum from one of the original pedigrees for Hp antibodies to determine whether Hp infection could explain the ulcers and elevated PG1 levels. METHODS: ELISA tests were performed using the urease fraction of a crushed Hp extract. Banked serum from one of the original families was thawed and tested. RESULTS: Of the subjects, 90% (nine of 10) with elevated PG1 were seropositive for Hp, compared to only 31% (17 of 55) of those with normal PG1 levels (p < 0.001). The mean PG1 level was higher in the seropositive (94.1+/-13.3 ng/ml) than the seronegative subjects (54.8+/-3.6, p < 0.05). Three of the four subjects with ulcers were Hp-seropositive. The prevalence of Hp-seropositivity and elevated PG1 declined in parallel in each successive generation. When neither parent was seropositive, children were seronegative. CONCLUSIONS: The etiology of elevated PG1 levels in this pedigree is more likely due to Helicobacter pylori infection than to a genetic predisposition.

Quantitative comparison on the refinement of horse antivenom by salt fractionation and ion-exchange chromatography.

A quantitative comparison was made on the fractionation of pepsin-digested horse antivenoms by ammonium sulfate (AS) fractional precipitation and ion-exchange chromatography on Q-Sepharose. In the precipitation process, pepsin digested horse anti-Naja kaouthia serum was precipitated by 30% saturated AS followed by 50% saturated AS. The recovery of antibody activity [as measured by an enzyme-linked immunosorbent assay (ELISA) against the cobra postsynaptic neurotoxin 3] from the 30-50% saturated AS precipitate was 53% with a 1.93-fold purification. For the chromatographic process, the behavior of the horse antitoxin antibody and its F(ab')2 fragments was first studied. The pepsin digested horse serum was then desalted on a Bio-gel P-2 column followed by chromatography on Q-Sepharose using a linear gradient (20 mM Tris-HCl, pH 8.0 containing 0.0 to 0.5 M NaCl) A peak containing primarily the F(ab')2 antibody could be obtained. This peak constituted 73% of the total antivenom activity with 2.08-fold purification. The total recovery of antibody activity by the chromatographic process was 90%. The yield of antibody activity was about 2-fold higher than that reported previously with other fractionation procedures. The implications of these results for the refining of horse therapeutic antivenoms are discussed.

[Characteristics of enzymatic function of the stomach in gastroduodenal diseases in children with regard to gastric carcinogenesis]. / Osobennosti fermentativnoi funktsii zheludka pri gastroduodenal'noi patologii u detei v aspekte gastrokantserogeneza.

The paper deals with a description of the biochemical properties of the isoforms of pepsinogen pepsin of gastric mucosa and blood serum in children suffering from duodenal ulcerative disease as well as in atrophic and subtrophic lesions of gastric mucosa. Atrophic gastritis was found to involve an inhibited biosynthesis of the Ist fraction of pepsinogen while ulcerative-erosive lesions of the gastro-duodenal area--an increased level of the 3rd isoform of pepsinogen.

Expression of interstitial collagenase, 92-kDa gelatinase, and tissue inhibitor of metalloproteinases-1 in granuloma annulare and necrobiosis lipoidica diabeticorum.

Granuloma annular (GA) and necrobiosis lipoidica diabeticorum (NLD) are disorders characterized by granulomatous inflammation and degenerative changes in collagen and elastic fibers. Because these disorders have often been described as being associated with altered extracellular matrix deposition, we studied the in situ expression of interstitial collagenase, 92-kDa gelatinase, and tissue inhibitor of metalloproteinases (TIMP)-1. Twelve lesions each of GA and NLD of different histopathologic types and durations were examined. Interstitial collagenase mRNA was seen in histiocyte-like cells in one-third of the cases of both diseases, typically in younger lesions. In GA, collagenase mRNA was only detected in lesions of the palisading type. Signal for 92-kDa gelatinase mRNA was observed in eosinophils, which were present in low numbers in five of 12 GA and three of 12 NLD samples. The signal for this enzyme and the presence of eosinophils did not correlate with the age of lesion. TIMP-1 mRNA was consistently expressed by histiocyte-like cells in both disorders. In GA, TIMP-1 mRNA was detected at the outer edge of the palisading granulomas, but in NLD, inhibitor expression was seen in the perivascular and periadnexal accumulation of inflammatory cells. Our data indicate that collagenase and TIMP are expressed early in these disorders and that these proteins may contribute to stromal remodeling associated with necrobiotic lesions. Our results further indicate that the localization of TIMP-1 production may provide a distinction between the two disorders, whereas metalloproteinase expression is not sufficiently specific to aid in the differential diagnosis of GA and NLD.

Doxycycline in the protection of serum alpha-1-antitrypsin from human neutrophil collagenase and gelatinase.

The concentration of doxycycline required to inhibit 50% (50% inhibitory concentration for serpinase activity) of alpha-1-antitrypsin degradation by purified neutrophil collagenase was found to be approximately 20 microM, a value similar to the 50% inhibitory concentration of doxycycline required to inhibit collagen degradation by neutrophil collagenase. Doxycycline also efficiently inhibited phorbol myristate acetate-triggered neutrophil-mediated degradation of alpha-1-antitrypsin. This suggests that doxycycline can protect alpha-1-antitrypsin from collagenase and gelatinase in the presence of other proteases and biologically active molecules that are released by triggered neutrophils. The protection of a body's alpha-1-antitrypsin shield from serpinolytic activity of collagenase and matrix metallproteinases can result in inhibition of serine proteases such as neutrophil elastase. Tetracyclines may thus protect matrix constituents from a wider spectrum of neutral proteases than previously recognized, not just from the matrix metalloproteinases collagenase and gelatinase.

Human neutrophil gelatinase is a collagenase type IV.

Secreted gelatinase from human neutrophils was purified as a 94 kDa polypeptide. Gelatinolytic and type IV collagenolytic activities of the purified protein were measured and compared. Immunoglobulins purified from antisera raised against gelatinase inhibited both the gelatinase and type IV collagenase activities. There was no cross-reaction in the inhibition with type I collagenase while the three metalloproteases were similarly inhibited by recombinant tissue inhibitor of metalloproteases. Purified gelatinase degraded denatured type I and native type IV collagens; there was no proteolysis of native type I collagen.

Gastric anti-ulcer activity of silymarin, a lipoxygenase inhibitor, in rats.

Oral treatment with silymarin was found to be effective in the prevention of gastric ulceration induced by cold-restraint stress, in rats. Statistically significant ulcer index values with respect to the control group, were observed. In 6 h pyloric-ligated animals silymarin showed a significant reduction in the number and severity of the ulcers; however, it did not alter the gastric secretion volume or acidity although histamine concentration was significantly decreased. In absolute ethanol-induced ulcers, treatment with silymarin 1 or 2 h before the anti-ulcerogenic agent, did not prevent the formation of gastric lesions. Furthermore, the hexosamine content was decreased significantly, but the total protein output was enhanced, showing similar values to those with the standard drug, carbenoxolone. These results suggest that the anti-ulcerogenic effect of silymarin could be related to its inhibitory mechanism of enzymatic peroxidation by the lipoxygenase pathway, avoiding leukotriene synthesis.

Subcellular localization and release of human neutrophil gelatinase, confirming the existence of separate gelatinase-containing granules.

An e.l.i.s.a. was developed using specific polyclonal rabbit antibodies against human neutrophil gelatinase. This assay, in contrast to the functional assay, is independent of activation of gelatinase, and is specific for the detection of gelatinase in both its reduced and unreduced forms. Using this assay, we were able to demonstrate a difference between the subcellular localization of gelatinase on the one hand, and the subcellular localization of vitamin B-12-binding protein, lactoferrin and cytochrome b558 on the other hand. The latter three co-localized in fractions of slightly higher density than gelatinase on a two-layer Percoll density gradient. Furthermore, the release of gelatinase exceeded the release of vitamin B-12-binding protein as well as lactoferrin by a factor of 3-6 following stimulation with formylmethionyl-leucyl-phenylalanine, leukotriene B4 and other soluble stimuli. Thus, although gelatinase has previously been found to co-localize with lactoferrin on immuno-electron microscopy, we confirm the existence of gelatinase-rich and lactoferrin- and vitamin B-12-binding-protein-poor granules, that are lighter and mobilized more easily than specific granules. These gelatinase-containing granules are not the store of cytochrome b558.

Simultaneous mobilization of Mac-1 (CD11b/CD18) and formyl peptide chemoattractant receptors in human neutrophils.

Mobilization of a distinct subset of specific granules provides a physiologically important mechanism to recruit Mac-1 (CD11b/CD18) from an intracellular pool to the external surface of the neutrophil plasma membrane, where the functionally active heterodimer mediates several adherence-dependent processes that are crucial for adequate host defense and cellular inflammatory responses. We observed similar characteristics for translocation of Mac-1 and neutrophil formyl peptide receptors (FPR) and hypothesize that the readily accessible pools of both Mac-1 and FPR are colocalized within this specific granule subset. Plasma membrane levels of both FPR (assessed with 3H-FMLP) and Mac-1 (assessed by fluorescence-activated cell sorter analysis of fluorescein isothiocyanate [FITC]-Mo-1-labeled cells) were markedly downregulated in cells prepared at low temperature from blood cooled to 4 degrees C immediately after removal from the circulation. Levels of both FPR and Mac-1 remained low on cells held at 4 degrees C. Upon warming, spontaneous upregulation of Mac-1 and FPR occurred with similar kinetics and temperature dependency. Translocation of both Mac-1 and FPR was markedly potentiated by exposure of cells to either fluoride ion (which has been shown by others to specifically elicit exocytosis of gelatinase-rich and vitamin B-12 binding protein-poor granules) or granulocyte-macrophage colony-stimulating factor (GM-CSF), a cytokine that markedly potentiates the neutrophils' host defense capabilities. Levels of both FPR and Mac-1 on F-- or GM-CSF-treated neutrophils exceeded those present on cells incubated at 37 degrees C for extended time intervals, indicating that stimulated translocation may involve mobilization of an additional granule subset. Scatchard analysis showed that only low-affinity FPR were translocated during spontaneous and stimulus-dependent upregulation. To directly compare FPR levels on the surface of cells displaying varying levels of Mac-1 within a single cell suspension, cells were labeled with FITC-Mo-1 and sorted into subpopulations based on fluorescence intensity. After sorting, the individual populations were held at 4 degrees C to prevent further spontaneous upregulation, concentrated by centrifugation, and assayed for FPR levels. Under a variety of conditions, FPR levels correlated with Mac-1 (CD11b) expression on cell populations selected on the basis of CD11b fluorescence intensity. Analysis of subcellular fractions obtained from disrupted neutrophils before and after upregulation provided additional support for the hypothesis that Mac-1 and FPR are colocalized within a readily accessible subset of neutrophil granules.(ABSTRACT TRUNCATED AT 400 WORDS)

Separation of fibronectin from a plasma gelatinase using immobilized metal affinity chromatography.

Conventional preparations of plasma fibronectin are known to contain a co-purifying gelatinase [1986, J. Biol. Chem. 261, 4363-4366], but so far useful methods to remove the protease have not been available. In this study a number of different methods were tested in order to achieve separation of the two proteins. Immobilized metal affinity chromatography was found to be efficient for this purpose, and a convenient procedure to separate the two proteins under nondenaturing conditions on chelating Sepharose charged with Co2+, Ni2+, or Zn2+ is described. An alternative method employing pH gradient elution of an Fe3+ gel also resolved fibronectin from the gelatinase. The Fe3+ gel bound both proteins at pH 6.0 but not at pH 7.4, suggesting that the two proteins were phosphorylated. The described procedures will now allow studies of the functions of fibronectin in the absence of the contaminating protease.

Parathyroid hormone regulation of matrix degrading enzymes in rat osteoblastic osteosarcoma 17/2.8 cells.

The present study was designed to further understand the role of PTH on the secretion of the neutral metalloproteinases, collagenase and gelatinase, from the rat osteosarcoma clonal cell line, ROS 17/2.8. Semiconfluent cells were treated with bovine parathyroid hormone, b-PTH-(1-34) at 100 nM-0.01 nM for 24-96 hours and pooled, concentrated media were analyzed by functional assay for collagenase (3H-methyl collagen) and gelatinase (3H-methyl gelatin). Collagenase activity significantly decreased (P less than 0.01) in the PTH conditioned media in a dose-dependent manner before (98-64%) and after (91-39%) reduction and alkylation. SDS-PAGE and fluorography apparently showed the most degradation to alpha A chains in collagen with controls, whereas this substrate remained intact with PTH (100 nM). PTH (100 nM) media also showed neutral gelatinase activity approximately 2% compared to control before and after reduction and alkylation (P less than 0.01). Significant amounts of an inhibitor to collagenase and gelatinase might have been secreted at 1 nM and 0.01 nM PTH, since collagenase and gelatinase activities were greater after reduction and alkylation. Reduction and alkylation likely destroyed these significant amounts of inhibitor. Polymorphonuclear leukocyte collagenase activity was also inhibited 80% by PTH conditioned media, but not by control. However, upon reduction and alkylation which destroyed inhibitor, the PTH treated media showed only a 14% inhibition against polymorphonuclear leukocyte collagenase (P less than 0.01). PTH appeared to downregulate neutral metalloproteinase activities through its effects on an inhibitor. This downregulation may represent a specific phenotypic response to PTH in ROS 17/2.8 cells.

Different effects of hypochlorous acid on human neutrophil metalloproteinases: activation of collagenase and inactivation of collagenase and gelatinase.

Human neutrophils stimulated with phorbol 12-myristate 13-acetate (PMA) produce the reactive oxidant hypochlorous acid (HOCl) and release the matrix metalloproteinases collagenase and gelatinase from secretory granules. We have investigated the stoichiometry of activation and inactivation of the two metalloproteinases with HOCl. HOCl activated purified neutrophil procollagenase at ratios between 10 and 40 mol of HOCl/mol enzyme, but caused inactivation at higher ratios. Maximum activation was about the same as that achieved by p-aminophenyl-mercuric acetate. However, less than a third of the total collagenase released from PMA-stimulated neutrophils was activated by coreleased HOCl and most of the activity was destroyed after 1 h of stimulation. These results indicate that the HOCl/enzyme ratio must fall within a narrow range for activation to occur. In contrast to collagenase, purified progelatinase underwent negligible activation (2.5 +/- 1.2%) at HOCl/enzyme molar ratios less than 30 and was destroyed at higher ratios. Likewise no active gelatinase could be detected in supernatant from PMA-stimulated cells and almost all of the proenzyme was destroyed by HOCl after 60 min stimulation. Our results illustrate that only collagenase can be activated by HOCl in vitro and that gelatinase is much more sensitive to inactivation. Since a precise HOCl/enzyme ratio is required for collagenase activation it is doubtful whether effective enzyme regulation by HOCl could occur in vivo where various HOCl scavengers are present.

Vomit identification by a pepsin assay using a fibrin blue-agarose gel plate.

Reported is a simple and reliable method for identifying the presence of gastric fluid in forensic samples by an assay that reveals the pepsin activity. These samples are usually vomit found at the scene of a crime, either in fresh form or as a dried stain on clothing. The pepsin within the sample is assayed for its proteolytic activity which is revealed in a fibrin blue-agarose gel plate, as a result of an enzymatic reactivity that takes the form of a concentric, blue, translucent ring around the tested sample. Apart from being able to determine the pepsin content of fresh or recent forensic samples, this method has also achieved positive reactions in aged gastric fluid stains that were kept at room temperature. No body fluids other than the gastric fluid and no proteolytic enzymes other than pepsin show a positive reaction with the use of this method. This method has an additional advantage, in that the enzymatic activity seen on the gel plate can be photographed and the gel plate, on drying, can also be preserved as evidence.