New monoclonal antibodies against gastric gland mucous cell-type mucins: a comparative immunohistochemical study

 The immunohistochemical reactivity of monoclonal antibodies raised against rat and pig gastric mucins (HIK1083, PGM36, and PGM37) was investigated in normal gastrointestinal tracts obtained from fish, amphibians, reptiles, birds, and mammals (including humans). These monoclonal antibodies exhibited highly selective reactivity with class III mucins, as identified by paradoxical concanavalin A stain, in the gastrointestinal tract of vertebrates. All three monoclonal antibodies reacted with the mucous neck cells and pyloric gland cells of amphibians, reptiles and mammals, the cardiac glands of reptiles and mammals, and Brunner’s glands of mammls. The deep crypt secretory cells of the rat colon and certain goblet-type cells deep in crypts in the pig colon differed from the above pattern only in that they did not show immunoreactivity with monoclonal antibody PGM36. These data suggest that the development of class III mucin is a fundamental evolutionary characteristic of vertebrate gastric mucins. These monoclonal antibodies should prove useful for the investigation of cell differentiation among gastrointestinal mucous cells and for the biochemical analysis of gastrointestinal mucins in different species. Accepted: 17 February 1998     

Helicobacter pylori infection up-regulates gland mucous cell-type mucins in gastric pyloric mucosa

Background. Two types of mucous cell are present in gastric mucosa: surface mucous cells (SMCs) and gland mucous cells (GMCs), which consist of cardiac gland cells, mucous neck cells, and pyloric gland cells. We have previously reported that the patterns of glycosylation of SMC mucins are reversibly altered by Helicobacter pylori infection. In this study, we evaluated the effects of H. pylori infection on the expression of GMC mucins in pyloric gland cells. Methods. Gastric biopsy specimens from the antrums of 30 H. pylori‐infected patients before and after eradication of H. pylori and 10 normal uninfected volunteers were examined by immunostaining for MUC6 (a core protein of GMC mucins), α1,4‐N‐acetyl‐glucosaminyl transferase (α4GnT) (the glycosyltransferase which forms GlcNAcα1‐4Galβ‐R), and GlcNAcα1‐4Galβ‐R (a GMC mucin‐specific glycan). Results. MUC6, α4GnT, and HIK1083‐reactive glycan were expressed in the cytoplasm, supranuclear region, and secretory granules in pyloric gland cells, respectively. The immunoreactivity of MUC6 and α4GnT, but not of GlcNAcα1‐4Galβ‐R, in the pyloric gland increased in H. pylori‐associated gastritis, and after the eradication of H. pylori, the increased expression of MUC6 and α4GnT in the gastric mucosa of H. pylori‐infected patients decreased to almost normal levels. This up‐regulation was correlated with the degree of inflammation. Conclusions. In addition to the synthesis of GMC mucins increasing reversibly, their metabolism or release may also increase reversibly in H. pylori‐associated gastritis. The up‐regulation of the expression of gastric GMC mucins may be involved in defense against H. pylori infection in the gastric surface mucous gel layer and on the gastric mucosa.     

Coexistence of gland mucous cell-type mucin and lysozyme in gastric gland mucous cells

Class III mucin, identified by paradoxical concanavalin A staining, is confined to gastric gland mucous cells and is an essential component of the gastric surface mucous gel layer. The pretreatment required has hampered the application of this method to electron microscopic studies. Antibody HIK1083 reacts selectively with class III mucins. The present study was undertaken to explore, electron microscopically, the immunoreactivity of the human stomach to HIK1083. We examined normal mucosa from resected human stomachs (five cases; formalin-fixed, paraffin-embedded) and gastric biopsy specimens from patients with early gastric cancer [nine cases; glutaraldehyde- and osmium-fixed, epoxy-embedded (seven cases) and half-strength Karnovsky’s solution-fixed, Lowicryl K4M-embedded (two cases)]. Immunostaining with HIK1083 and anti-lysozyme antibody was examined under light and electron microscopes. Gland mucous cells were labeled with HIK1083, and lysozyme was detected in some gland mucous cells and surface mucous cells. Electron microscopically, the secretory granules of gland mucous cells contained a single electron-dense core. HIK1083-positive mucins and lysozyme coexisted in the secretory granules of gastric gland mucous cells. HIK1083-reactive mucins and lysozyme were distributed in the matrix and in the dense core of these secretory granules, respectively. HIK1083 can be used for electron immunohistochemistry. Accepted: 1 December 1999     

Rat gastric mucins recognized by monoclonal antibodies RGM21 and HIK1083: isolation of mucin species characteristic of the surface and glandular mucosa.

Whole mucins and reduced subunits were extracted from the corpus of the rat stomach. After purification by Sepharose CL-4B chromatography followed by cesium trifluoroacetate equilibrium centrifugation, they were analyzed by Sepharose CL-2B chromatography, rate-zonal sedimentation centrifugation, and Q-Sepharose chromatography. Monoclonal antibodies RGM21 and HIK1083, which histochemically stained mucins in the surface and glandular mucosa of the rat stomach, respectively, were used to detect the site-specific mucins. Although RGM21- and HIK1083-reactive mucins both had a multimerized structure, the density and size of both the whole mucins and reduced subunits differed, thus indicating the presence of distinct mucin species in the surface and glandular mucosa. The mucin subunits were separated into four fractions, UB, B1, B2a, and B2b, by Q-Sepharose chromatography. HIK1083 reacted mainly with UB, while RGM21 reacted with B1, B2a, and B2b. These results, combined with dot-blot, amino acid, and carbohydrate composition analyses, showed that the surface mucins may consist of three kinds of subunits. In contrast, the glandular mucins may consist of one kind of subunit which differs from that of surface mucins.     

Characterization of a CD3-like rat T cell surface antigen recognized by a monoclonal antibody

A mouse mAb, which recognized a rat T cell surface Ag responsible for the T cell activation, was produced by a regular hybridoma method using F344 rat T cells stimulated with PMA and a calcium ionophore, as the Ag. The mAb termed 1F4 (kappa-IgM) was reactive with rat T cells but not with B cells and immunohistochemically it stained rat thymus tissues strongly at medulla and weakly rat cortex. Addition of 1F4 mAb to a culture of T cells resulted in the proliferation of T cells by a help of PMA or a solid support. 1F4 mAb also caused the modulation of the corresponding Ag but not other T cell markers such as CD5, CD2, and OX-52-defined Ag. The 1F4 mAb immunoprecipitated a cell surface component having an apparent m.w. of 25,000 from rat T cells which could be associated with a disulfide-linked heterodimer (m.w. 92,000) consists of subunits having m.w. of about 52,000 and 43,000. These results strongly suggest that the 1F4 mAb recognizes a rat T cell Ag homologous to the human and mouse CD3.     

Characterization of rat prothymocyte with monoclonal antibodies recognizing rat lymphocyte membrane antigenic determinants

Utilizing the technique of fluorescence-activated cell sorting and monoclonal antibodies directed at rat membrane antigens, various subpopulations of Lewis bone marrow cells were isolated and subsequently transfused into sublethally irradiated, histocompatible NBr recipient rats by either intravenous or intrathymic inoculation. Recipient rats were sacrificed and cell suspensions from thymus and other lymphoid tissue were examined for the presence of the RT7.1 marker on Lewis thymus-derived lymphocytes by fluorescence-activated cell analysis. From these studies, the population of Lewis bone marrow cells that could reconstitute T cells in the NBr rats was found to be Ox-22 negative, Ox-7 positive, W3/13 positive, and Ox-18 positive. Further analysis characterized the prothymocyte as being Ox-7 upper 20% positive and W3/13 weakly positive. In addition this marrow cell population was able to protect lethally irradiated Lewis rats (9.5 Gy) in 30-day survival tests. These studies have indicated that the prothymocyte either has been derived from the Ox-22 negative, Ox-7 upper 20% positive, and W3/13 positive marrow cells or, like the hematopoietic stem cell, this cell has also been characterized by this phenotype.     

MZ15, a monoclonal antibody recognizing keratan sulphate, stains chick tendon

Summary Biochemical, morphometric and electron histochemical methods have failed to demonstrate the presence in tendon of keratan sulphate, a common component of connective tissue proteoglycans. Using a monoclonal antibody specific for keratan sulphate, a positive localization of this molecule was made in the gastrocnemius tendon of stage 44 chicken embryos both at the light and electron microscopical levels.     

Actions of a monoclonal antibody Tor 23 on rat brain presynaptic cholinergic processes

Tor 23 is a monoclonal antibody, generated against cholinergic terminals of theTorpedo californica, that has been found to bind to the extracellular surface of cholinergic neurons in a variety of tissues. This study shows that Tor 23 inhibits: 1) high affinity [³H]hemicholinium-3 binding to detergent-solubilized membranes prepared from rat neocortices; 2) high affinity [³H]choline uptake in rat neocortical and striatal P₂ preparations; and 3) [³H]acetylcholine synthesis in isolated nerve terminals. Tor 23 does not appear to affect low affinity [³H]choline uptake or [³H]acetylcholine release. These results are consistent with the hypothesis that Tor 23 may bind to nerve terminal high affinity choline transporters in the rat brain.     

A novel monoclonal antibody recognizing a unique antigen of rat osteoclasts induced by the calcified matrices

The morphology of osteoclasts, primary cells that resorb bone, is well documented; however, the precise details of their terminal differentiation remains obscure. To date, the only morphological criterion for identifying activated functional osteoclasts has been the presence of ruffled borders. We have developed a rat bone marrow culture system in which osteoclast-like cells formed. These cells fulfilled most of the criteria of osteoclasts, and when they were reseeded on calcified tissue, formed numerous resorption lacunae in vitro. To find an immunological marker for functional osteoclasts, we have used these cells in a functional state as antigens for the preparation of monoclonal antibodies (mAb) that reacted with rat osteoclasts; we obtained mAb Ch1 and Ch2. Interestingly, these mAbs reacted with the marginal portion of authentic osteoclasts, where they attached to the bone surface on frozen sections. The reactivity of Ch1 to rat osteoclasts was more restricted than that of Ch2: Ch1 reacted with few tartrate-resistant acid phosphatase (TRAP)-positive cells on a culture plate. These TRAP-positive cells (including mono- and multinucleated cells) were, however, converted to Ch1-positive cells when they were reseeded on calcified tissues. These findings suggested that the antigen recognized by the Ch1 antibody was induced by some factors of matrix proteins released from calcified tissues.     

Characterization of rat OX40 ligand by monoclonal antibody

OX40 (CD134) is a member of the tumor necrosis factor (TNF) receptor superfamily first identified as a rat T cell activation marker. We previously identified the rat ligand for OX40 (OX40L) by molecular cloning. In the present study, we newly generated an anti-rat OX40L mAb (ATM-2) that can inhibit the binding of OX40 to rat OX40L and thus efficiently inhibits the T cell costimulatory activity of rat OX40L. Flow cytometric analyses using ATM-2 and an anti-rat OX40 mAb (MRC OX40) indicated that OX40 was inducible on splenic CD4(+) T cells by stimulation with immobilized anti-CD3 mAb, while OX40L was not expressed on resting or activated T cells. OX40L was expressed on splenic B cells after stimulation with lipopolysaccharide (LPS), but not on peritoneal macrophages. Interestingly, splenic dendritic cells (DC) expressed OX40L constitutively, which was further upregulated by LPS stimulation. The potent costimulatory activities of splenic DC for anti-CD3-stimulated rat CD4(+) T cell proliferation and cytokine (IL-2, IFN-gamma, IL-10, and IL-13) production were substantially inhibited by ATM-2. These results indicated that OX40L is expressed on professional antigen-presenting cells (APC), and may be involved in humoral immune responses via T-B interaction and in cellular immune responses via T-DC interaction in the rat system.     

Immune deposits formed in situ by a monoclonal antibody recognizing a new intrinsic rat mesangial matrix antigen

We describe a unique mesangial matrix component of the rat glomerulus identified by a murine monoclonal antibody. The antigen is present exclusively in the glomerular mesangium and cannot be detected in other rat tissues by indirect immunofluorescence techniques or following pretreatment of tissue sections with acid urea or other nonionic detergents. Specific immunoprecipitation of the solubilized antigen yields a single peptide with an apparent m.w. of 81,000 when analyzed by discontinuous SDS-PAGE. This mesangial matrix component is collagenase resistant and trypsin sensitive. Perfusion of an isolated kidney preparation with this antibody results in direct binding of the mouse immunoglobulin to its mesangial antigen. Passive administration of the monoclonal antibody to Lewis rats results in characteristic electron dense deposits within the mesangial matrix that can be visualized ultrastructurally as early as 3 days. The immune deposits form without the activation of rat complement and persist for longer periods than those that develop after the planting of aggregated proteins or preformed immune complexes. Experimental animals that received either a monoclonal antibody specific for laminin or a non-kidney binding preparation did not develop such immune deposits at any time during the course of the autologous phase of the immune process. The results obtained in this study indicate that electron dense immune deposits can develop in the mesangium with the participation of a unique intrinsic matrix component and specific circulating monoclonal antibodies by an in situ mechanism of immune complex formation.     

Characterization of a monoclonal antibody, HTA28, recognizing a histone H3 phosphorylation site as a useful marker of M-phase cells.

Mitosis is a valuable indicator of active tissue proliferation but, other than morphological characteristics, there have hitherto been no markers available to detect only M-phase cells. However, a newly established monoclonal antibody (MAb), HTA28, recognizing histone H3 (H3) harboring phosphoserine 28, allows visualization with mitotic chromosomal condensation. In this study we investigated the use of HTA28 for immunohistochemical (IHC) detection of M-phase cells in the regenerating rat liver after partial hepatectomy (PH). Groups of three to five rats were sacrificed at intervals up to 72 hr after PH and proliferation was then assessed by IHC staining using HTA28 and other markers. The temporal pattern of the HTA28 staining index (SI) was very similar to that for the mitotic index (MI), also showing similarities to the bromodeoxyuridine (BrdU) labeling index (LI) with a time lag. The HTA28 SI proved to be higher than MI at every time point in line with HTA28 immunoreactivity maintained for all stages of M-phase. The spatial distribution of HTA28-positive cells corresponded with those of other proliferative cell markers. These therefore provide strong evidence for the applicability of HTA28 as an M-phase marker. We also showed that antigenicity for HTA28 is lost if tissue is not immediately fixed after sampling.     

Characterization of Yersinia pestis fibrinolysin using monoclonal antibody]

Immunochemical identity of Y. pestis fibrinolysin and coagulase is demonstrated using monoclonal antibodies. These substances are proven to exist as complex proteins with two independent activities. Possible causes of this phenomenon are discussed. Coagulase antigenic determinants are involved in specific fluorescence of Y. pestis cells grown at 28 degrees C. A new original method for screening the hybridomas producing monoclonal antibodies is proposed, based on inhibition of the functional activity of antigen.     

Enriched populations of rat retinal ganglion cells: Studies using a cell-type specific surface marker

Cells dissociated from adult and neonatal rat retinas were separated by density gradient centrifugation. Previous work had shown that rat retinal cells labelled by an immunofluorescence assay for the Thy-1 antigen were chiefly or exclusively ganglion cells, and so the proportion of Thy-1 positive cells in the density gradient fractions was used as an index of the enrichment of ganglion cells. The proportion of Thy-1 positive neonatal cells was increased from about 0.4% in the initial dissociate to about 8% in the most enriched fraction of a Percoll step gradient. Amongst adult cells the initial 0.7% Thy-1 positive cells were increased to roughly 2% in the best fraction of a metrizamide step gradient.

The presence of relatively large numbers of Thy-1 positive cells in other fractions suggested that it would be difficult to further increase the proportion of rat ganglion cells by methods based on their sedimentation properties. These results demonstrate the importance of cell-type specific markers in attempts to purify cells from the central nervous system.     

Distribution of parvalbumin-immunoreactivity in the rat thalamus using a monoclonal antibody

1. The distribution of parvalbumin cell bodies and fibers in the thalamus of the rat was studied using a monoclonal antibody and the avidin-biotin-peroxidase method. The densest clusters of immunoreactive perikarya were observed in the nuclei ventralis posterior, reticularis, ventralis anterior and zona incerta, whereas the nuclei habenularis lateralis, lateralis posterior, lateralis, centralis lateralis and ventralis lateralis had the lowest density. In the nucleus geniculatum laterale ventralis, the density of parvalbumin cell bodies was intermediate. In all these thalamic nuclei, small, round or fusiform immunoreactive cells with short immunolabeled dendritic processes were observed. 2. The densest network of immunoreactive fibers was observed in the nuclei geniculatum laterale ventralis, reticularis and zona incerta. The nuclei geniculatum laterale dorsalis, ventralis posterior, medialis ventralis, ventralis anterior, anterior ventralis, anterior dorsalis and rhomboidens contained a moderate number of parvalbumin fibers, whereas the nuclei lateralis posterior, habenularis lateralis, parataenialis, centrum medianum, lateralis, centralis lateralis, ventralis lateralis, medialis dorsalis, anterior medialis, ventralis medialis and lateralis anterior had the lowest density of immunoreactive fibers. In addition, a large number of immunoreactive fibers was found in the lemniscus medialis and a scarce number in the stria medullaris. 3. No immunoreactive structure was observed in the nuclei habenularis medialis, paraventricularis, reuniens and geniculatum mediale. 4. Thus, perikarya and fibers containing parvalbumin are widely distributed throughout the thalamus of the rat, suggesting that parvalbumin might play a role, directly or indirectly, in limbic, visual and somatosensory mechanisms.     

Characteristics of a monoclonal beta-endorphin antibody recognizing the N-terminus of opioid peptides

The properties of a mouse monoclonal antibody to beta-endorphin secreted by a clone of hybrid myelomas (3-E7) are described. The antibody displays virtually complete cross-reactivity to met-enkephalin and leu-enkephalin, but no cross-reactivity to beta-lipotropin, alpha-N-acetyl-beta-endorphin and des-Tyr1-beta-endorphin. Substantial cross-reactivity is seen with some other naturally occurring opioid peptides bearing the enkephalin sequence, such as dynorphin, alpha-neo-endorphin and BAM 22, but cross-reactivity is lacking in the case of certain synthetic enkephalin derivatives possessing a D-amino acid in position 2. The data indicate that for the binding of an antigen to the antibody the N-terminal tyrosine moiety is essential. The antibody recognizes, thus, a site which is of functional significance for the interaction of many naturally occurring opioid peptides with the opiate receptor.     

A dual staining method for identifying mucins of different gastric epithelial mucous cells

Summary A dual staining method has been developed to identify two types of mucous secreting cells in the gastric mucosa of human and rat in one and the same tissue section. Sections were stained first using the galactose oxidase-cold thionin Schiff (GOCTS) procedure and then with paradoxical Concanavalin A staining (PCS). Surface mucous cell mucin stained blue with GOCTS, whereas gland mucous cell mucin stained brown with PCS. This method enabled us to differentiate these two types of mucins not only in gastric epithelial cell cytoplasm but also in the extracellular space. Sugar residues detected by GOCTS were explored by employing four species of lectins, which were peanut andAllomyrina dichotoma agglutinins for -galactose andVicia villosa andWistaria floribunda agglutinins for -N-acetylgalactosamine. The effect of oxidation with galactose oxidase was also examined on the affinities of reactive sites for these lectins. The results indicated that, in the human stomach, the sugar residues responsible for this reactivity were most likely -N-acetylgalactosamine and -galactose in specimens lacking secretion of blood group determinants and -N-acetylgalactosamine in those showing the secretion. In the rat stomach, on the other hand, sugar residues responsible for GOCTS were not elucidated by these lectins.