Cytochemical localization of alkaline phosphatase in intestinal metaplasia of the human stomach

Summary Alkaline phosphatase in the brush border of areas of intestinal metaplasia of human stomach was studied cytochemically. All absorptive cells in the upper part of the villi of the duodenum had strong alkaline phosphatase activity but, in areas of intestinal metaplasia, the metaplastic glands consisted of alkaline phosphatase-positive and negative absorptive cells. Alkaline phosphatase activity was found in tall dense microvilli of absorptive cells in areas of intestinal metaplasia and in the duodenum. However, in some areas of metaplastic epithelium, the activity was very weak in some tall dense microvilli of absorptive cells but strong in those of neighbouring absorptive cells. No alkaline phosphatase activity was found in short sparse microvilli of absorptive cells in areas of intestinal metaplasia. The difference in alkaline phosphatase activity in microvilli of different cells in areas of intestinal metaplasia, which is not seen in the duodenum, indicates abnormal morphological and enzymatic differentiation in intestinal metaplasia.     

潘氏细胞研究进展

潘氏细胞是位于小肠腺底部的浆液性腺上皮细胞,其主要特征是细胞顶部有大量粗大的嗜酸性分泌颗粒,内含防御素、溶菌酶、sIgA等多种抗菌物质。表达于潘氏细胞的NOD2、Toll样受体9、肝癌-肠-胰腺／胰腺炎相关蛋白、RegⅢγ、肿瘤坏死因子仅、粒细胞-巨噬细胞集落刺激因子、白介素-17等也是免疫与炎症反应的重要成分。金属硫蛋白、富半胱氨酸肠蛋白、潘氏细胞锌结合蛋白等金属结合蛋白均分布于潘氏细胞,提示潘氏细胞参与金属代谢。潘氏细胞是构成肠黏膜屏障的重要细胞成分。NOD2单核苷酸多态性与克罗恩病有关。潘氏细胞化生常发生于胃、大肠的炎症与肿瘤病变,其病理意义有待于进一步研究。     

3H]thymidine autoradiographic and alkaline phosphatase histochemical studies of intestinal metaplasia of the human stomach

The relationship between cell proliferation and enzyme activity in intestinal metaplasia of the human stomach was studied using a combined method of [3H]thymidine autoradiography and alkaline phosphatase histochemistry on the same section. Three types of intestinal metaplasia were observed depending on variations in both enzymatic activity and isotope labelling. One type shows alkaline phosphatase-positive cells along the entire length of the glands with [3H]thymidine-labelled cells localized only at the bottom of the glands, resembling the duodenum. In another type of intestinal metaplasia, alkaline phosphatase-positive cells are present on the surface and/or upper half of the glands with mitotically active cells occupying the lower part of the glands. The third variety of intestinal metaplasia is characterized by the absence of alkaline-phosphatase activity and [3H]thymidine-labelled cells present in an extended zone in the lower half of the glands. Differences in labelling patterns of [3H]thymidine and the activity of marker enzyme in various types of intestinal metaplasia seem to reflect variations in cell differentiation during intestinalization of gastric mucosa.     

A method of quantitating Paneth cell metaplasia of the stomach by image analysis

Paneth cells are one of the histologic components of intestinal metaplasia of the stomach, as are mucin-producing goblet cells. With the aid of an image quantifier, the distribution of Paneth cells histochemically labeled with acid fuchsin was analyzed for a gastrectomy specimen containing an adenocarcinoma of the intestinal type; the topographic distribution of goblet cells histochemically labeled with Alcian blue (pH 2.5) was also analyzed. The specimen was cut into 63 blocks (0.5 X 4.0 cm) in four zones; antrum (zone I), intermediate region (zone II) and fundus (zones III and IV). Paneth cells were found only in sections containing mucin-producing goblet cells. Paneth cells were found in 12.5% of the 16 sections from the antral zone I containing Alcian blue-positive goblet cells. The rates were 44.4% for the intermediate zone II and 55.5% for the distal fundic zone III. The total area occupied by Paneth cells was significantly lower in the gastric mucosa as compared to the duodenal mucosa. The "Paneth cell index" (total Paneth cell area/total goblet cell area) was highest in the duodenum, followed by the distal fundic zone III. This method of quantitating Paneth cell metaplasia of the stomach will be used to investigate the topographic distribution of those cells in populations with low and high incidences of intestinal metaplasia.     

Abnormal Paneth cell granule dissolution and compromised resistance to bacterial colonization in the intestine of CF mice

Paneth cells of intestinal crypts contribute to host defense by producing antimicrobial peptides that are packaged as granules for secretion into the crypt lumen. Here, we provide evidence using light and electron microscopy that postsecretory Paneth cell granules undergo limited dissolution and accumulate within the intestinal crypts of cystic fibrosis (CF) mice. On the basis of this finding, we evaluated bacterial colonization and expression of two major constituents of Paneth cells, i.e., alpha-defensins (cryptdins) and lysozyme, in CF murine intestine. Paneth cell granules accumulated in intestinal crypt lumens in both untreated CF mice with impending intestinal obstruction and in CF mice treated with an osmotic laxative that prevented overt clinical symptoms and mucus accretion. Ultrastructure studies indicated little change in granule morphology within mucus casts, whereas granules in laxative-treated mice appear to undergo limited dissolution. Protein extracts from CF intestine had increased levels of processed cryptdins compared with those from wild-type (WT) littermates. Nonetheless, colonization with aerobic bacteria species was not diminished in the CF intestine and oral challenge with a cryptdin-sensitive enteric pathogen, Salmonella typhimurium, resulted in greater colonization of CF compared with WT intestine. Modest downregulation of cryptdin and lysozyme mRNA in CF intestine was shown by microarray analysis, real-time quantitative PCR, and Northern blot analysis. Based on these findings, we conclude that antimicrobial peptide activity in CF mouse intestine is compromised by inadequate dissolution of Paneth cell granules within the crypt lumens.     

Use of Gallocyanin as a Myelin Stain for Brain and Spinal Cord

Tissue fixed in 10% formalin, formol saline, CaCO₃ or phosphate buffer neutralized formalin, Baker's formol calcium, Cajal's formol ammonium bromide, formalin-95% ethanol 1:9, formalin-methanol 1:9, Lillie's methanol-chloroform or Salthouse's formol cetyltrimethylammonium bromide was dehydrated and embedded in paraffin. Sections were attached to slides with either albumen or gelatine adhesive and processed throughout at room temperature of 22-25 C. Mordanting 30-60 min in 1% iron alum was followed by a 10 min wash in 4 changes of distilled water. Myelin was stained in a gallocyanin self-differentiating solution for 1-2.5 hr; thick sections requiring the longer time. The staining solution (pH approximately 7.4) consisted of Na₂CO₃, 90 mg; distilled water, 100 ml; gallocyanin, 250 mg; and ethanol, 5 ml. The ethanol was added to this mixture last, and after the other ingredients had been boiled and then cooled to room temperature. After a staining and thorough washing, Nissl granules were stained for 5-10 min in a solution consisting of: 0.1 M acetic acid, 60 ml; 0.1 M sodium acetate, 40 ml; methyl green, 500 mg. Washing, dehydration, clearing and mounting completed the process. Myelin sheaths were stained dark violet; neuronal nuclei, light green with dark granules of chromatin; nucleoli of motor cells and erythrocytes, dark violet; cytoplasm, green with dark green Nissl granules. The simple and reliable method can be adapted easily for use with automatic tissue processors.     

Utility of human placental alkaline phosphatase as a genetic marker for cell tracking in bone and cartilage

It was the aim of the current study to evaluate the utility of human placental alkaline phosphatase (hPLAP) as a genetic marker for cell tracking in bone and cartilage, using transgenic Fischer 344 rats expressing hPLAP under the control of the ubiquitous R26 promoter [F344-Tg(R26-hPLAP)]. hPLAP enzyme activity was retained during paraffin and methylmethacrylate (MMA) embedding, and was best preserved using 40% ethanol as fixative. Endogenous alkaline phosphatase activity could be completely blocked by heat inactivation in paraffin and MMA sections, allowing histochemical detection of hPLAP in the complete absence of background staining. In addition, sensitive detection of hPLAP was also possible using immunohistochemistry. F344-Tg(R26-hPLAP) rats demonstrated ubiquitous expression of hPLAP in hematopoietic bone marrow cells and stromal cells such as osteoblasts, osteocytes, and chondrocytes. Osteoclasts only weakly expressed hPLAP. In conclusion, hPLAP provides superb detection quality in paraffin and plastic sections, and constitutes an excellent genetic marker for cell tracking in hard and soft tissues.     

Association of Matrix Acid and Alkaline Phosphatases with Mineralization of Cartilage and Endochondral Bone

The activities of acid and alkaline phosphatases were localized by enzyme histochemistry in the chondroepiphyses of 5 week old rabbits. Using paraformaldehyde-lysine-periodate as fixative, the activity of acid phosphatase was particularly well preserved and could be demonstrated not only in osteoclasts, but also in chondrocytes as well as in the cartilage and early endochondral matrices. The acid phosphatase in the chondrocytes and the matrix was tartrate-resistant, but inhibited by 2mM sodium fluoride, whereas for osteoclasts 50–100mM sodium fluoride were required for inhibition. Simultaneous localisation of both acid and alkaline phosphatase activities was possible in tissue that had been fixed in 85% ethanol and processed immediately. In the growth plates of the secondary ossification centre and the physis, there was a sequential localisation of the two phosphatases associated with chondrocyte maturation. The matrix surrounding immature epiphyseal chondrocytes or resting/proliferating growth plate chondrocytes contained weak acid phosphatase activity. Maturing chondrocytes were positive for alkaline phosphatase which spread to the matrix in the pre-mineralising zone, in a pattern that was consistent with the known location of matrix vesicles. The region of strong alkaline phosphatase activity was the precise region where acid phosphatase activity was reduced. With the onset of cartilage calcification, alkaline phosphatase activity disappeared, but strong acid phosphatase activity was found in close association with the early mineral deposition. Acid phosphatase activity was also present in the matrix of the endochondral bone, but was only found in early spicules which had recently mineralised. The results suggest that alkaline phosphatase activity is required in preparation of mineralization, whereas acid phosphatase activity might have a contributory role during the early progression of mineral formation.     

The role of Paneth cells and their antimicrobial peptides in innate host defense

The intestinal epithelium is the largest surface area that is exposed to various pathogens in the environment, however, in contrast to the colon the number of bacteria that colonize the small intestine is extremely low. Paneth cells, one of four major epithelial cell lineages in the small intestine, reside at the base of the crypts and have apically oriented secretory granules. These granules contain high levels of antimicrobial peptides that belong to the alpha-defensin family. Paneth cells secrete these microbicidal granules that contain alpha-defensins when exposed ex vivo to bacteria or their antigens, and recent evidence reveals that antimicrobial peptides, particularly alpha-defensins, that are present in Paneth cells contribute to intestinal innate host defense.     

Immunocytochemical localization of pancreatic carboxylic ester hydrolase in human paneth cells

The protein-A gold method using specific rabbit sera directed against pure human pancreatic chymotrypsinogen and carboxylic ester hydrolase was applied to locate these (pro)enzymes in human pancreatic acinar cells and intestinal Paneth cells. Quantitative evaluation of the labelling indicated that both (pro)enzymes are present in pancreatic acinar secretory granules. In Paneth cell secretory granules, only carboxylic ester hydrolase was present in significant amounts, although the labelling for this enzyme was less intense than that observed in pancreatic zymogen granules. The results obtained support the view that Paneth cells represent a "diffuse exocrine gland" scattered along the intestine, whose role is either to act as a substitute in the event of a deficient pancreas or to regulate the intestinal flora.     

Ultrastructural localization of osteopontin immunoreactivity in phagolys osomes and secretory granules of cells in human intestine

A post-embedding ultrastructural immunogold method was used to detect osteopontin in human intestinal biopsies with special emphasis on secretory and phagocytic organelles. Osteopontin immunoreactivity was localized to phagolysosomes of macrophages, fibroblasts, absorptive epithelial cells of the small intestine and Paneth cells. The mucigen secretory granules and Golgi structures of mucous epithelial cells of the small intestinal epithelium contained osteopontin, but secretory granules of numerous other cells, including Paneth cells, did not. Extracellular and phagocytosed Tropheryma whippelii within macrophage phagolysosomes also bound osteopontin. These localizations are supportive of a role for osteopontin in phagocytic and some secretory cell functions in human intestine     

Functional Role of Metaplastic Paneth Cell Defensins in Helicobacter pylori-Infected Stomach

Background and Aims:  Chronic gastritis is caused by Helicobacter pylori infection, and gastritis is classified as inflammation, atrophy, and intestinal metaplasia. Detailed pathologic studies have shown that H. pylori settles on the surface of gastric mucosa, and that it is eliminated from metaplastic mucosa. However, its mechanism of natural protection is not well known.
Methods:  Antimicrobial human enteric defensin expression was determined in the RNA and protein levels. Recombinant enteric defensins were produced with a bacterial expression system and their anti- H. pylori activities were assessed by bactericidal assay.
Results:  Human enteric defensin (HD)-5 and HD-6 were detected in Paneth cells, which are observed in the gastric metaplastic mucosa as well as small intestinal epithelia. HD-5 protein was coexpressed with trypsin, which is considered to be an activating enzyme of HD-5. Less H. pylori was observed in the intestinal metaplasia with HD-5 expressing Paneth cells. The recombinant defensins showed killing activity against H. pylori at a low concentration in vitro.
Conclusions:  The human defensins that are expressed in the metaplastic Paneth cells eliminate H. pylori . Metaplastic change may be a purposive development of the human stomach.     

myo-inositol action on gerbil intestine: alterations in alkaline phosphatase activity upon phosphatidylinositol depletion and repletion in vivo

The influence of phosphatidylinositol (PI) on intestinal alkaline phosphatase activity was studied in myo-inositol deficient gerbils. A reduction of membrane PI in intestinal mucosa to 30-40% of the control was produced by feeding female gerbils a myo-inositol-deficient diet containing coconut oil for 2 weeks. As expected, the animals developed typical intestinal lipodystrophy with abnormal fat accumulation. In the PI-depleted animal, intestinal alkaline phosphatase activity was reduced to 20-30% of the control group. The levels of both membranous and soluble enzymes in intestinal mucosa were affected, but there were no changes in liver, kidney and plasma levels. When the lipodystrophic gerbils were given dietary myo-inositol, the complete repletion of intestinal membrane PI to the control level occurred 36 h later, whereas membrane-bound alkaline phosphatase activity in intestine was not restored to the control level until 72 h later. Administration of cycloheximide or actinomycin D did not block this enzyme induction. Lymphatic output of triacylglycerol into the bloodstream was stimulated 10-fold at 18 h of myo-inositol repletion, but there was no parallel increase in the activity of alkaline phosphatase in plasma during this early phase of intestinal recovery. Thus, these data suggest a possible regulatory role of PI in the processing and/or turnover of alkaline phosphatase in vivo, but a negative role of alkaline phosphatase in lipid transport across gerbil intestine.     

Histophysiology of digestion and observations on the structure of the alimentary canal in the ectosymbiont Chaetogaster limnaei limnaei Baer, 1827 (Annelida: Oligochaeta)

The naidid oligochaete Chaetogaster limnaei limnaei has an alimentary canal consisting of a mouth, pharynx with a dorsal pharyngeal pad, esophagus, stomach, anterior and posterior intestine, and anus. The diet is omnivorous but limited by particle size. Unattached food organisms are sucked into the pharynx while sessile organisms are plucked from the substratum. Granules of acid mucosubstances that stain purple with neutral red are secreted into the stomach lumen after food enters, rapidly increasing the acidity from pH 3 to 1.5. Acid induced lysis of the organisms initiates autolysis before the food is passed into the alkaline, pH 7 to 8, anterior intestine. Ciliated intestinal cells showed arylamidase, acid phosphatase and C-esterase active granules indicating primary lysosomes with secondary lysosomes being recognized in electron micrographs suggesting intracellular digestion. Arylamidase and alkaline phosphatase activity appears in the intestinal margins during the alkaline phase of digestion. Scattered, pyramidal cells found only in the anterior intestine contain yellow refractile spheres. The spheres stain alcian blue pH 2.5 and bromophenol blue positive and exhibit a strong acid phosphatase activity all the time with A-esterase active granules surrounding them. Glycogen and lipids are stored mainly in the chlorogague cells. Many of the yellow refractile granules in the stomach and intestinal cells are bacteria.     

Ultrastructural localization of osteopontin immunoreactivity in phagolys osomes and secretory granules of cells in human intestine

A post-embedding ultrastructural immunogold method was used to detect osteopontin in human intestinal biopsies with special emphasis on secretory and phagocytic organelles. Osteopontin immunoreactivity was localized to phagolysosomes of macrophages, fibroblasts, absorptive epithelial cells of the small intestine and Paneth cells. The mucigen secretory granules and Golgi structures of mucous epithelial cells of the small intestinal epithelium contained osteopontin, but secretory granules of numerous other cells, including Paneth cells, did not. Extracellular and phagocytosed Tropheryma whippelii within macrophage phagolysosomes also bound osteopontin. These localizations are supportive of a role for osteopontin in phagocytic and some secretory cell functions in human intestine This revised version was published online in November 2006 with corrections to the Cover Date.     

Chemical inactivation of HIV on surfaces.

To assess whether alcohol and glutaraldehyde are effective disinfectants against dried HIV the virucidal effects of 70% alcohol (ethanol and industrial methylated spirit) and 1% and 2% alkaline glutaraldehyde were tested against cell associated and cell free HIV dried on to a surface. Virus stock (100 microliters) or 10,000 cultured C8166 T lymphocytes infected with HIV were dried onto sterile coverslips and immersed in 2% and 1% alkaline glutaraldehyde and 70% ethanol for 30 seconds and one, two, four, and 10 minutes, there being an additional time point of 20 minutes for cell free virus disinfected with 70% industrial methylated spirit. In addition, virus stock in neat serum was tested with 1% and 2% alkaline glutaraldehyde to see whether the fixative properties of glutaraldehyde impair its virucidal properties. Virus activity after disinfection was tested by incubating the coverslips (cell associated virus) or the coverslips and sonicated cell free virus with C8166 T lymphocytes. The lymphocytes were examined for the formation of syncytia and HIV antigens were assayed in the culture fluid. Both 2% and 1% alkaline glutaraldehyde inactivated cell free HIV within one minute; 2% alkaline glutaraldehyde also inactivated cell free virus in serum within two minutes, but a 1% solution was ineffective after 15 minutes'' immersion. Cell associated HIV was inactivated by 2% alkaline glutaraldehyde within two minutes. Seventy per cent industrial methylated spirit failed to inactivate cell free and cell associated HIV within 20 and 15 minutes, respectively, and 70% ethanol did not inactivate cell free virus within 10 minutes. Seventy per cent industrial methylated spirit and ethanol are not suitable for surface disinfection of HIV. Fresh 2% solutions of alkaline glutaraldehyde are effective, but care should be taken that they are not too dilute or have not become stale when used for disinfecting HIV associated with organic matter.     

Light microscopic localization of alkaline phosphatase in fetal bovine bone using immunoperoxidase and immunogold-silver staining procedures

We localized alkaline phosphatase in the metaphyses of fetal bovine tibial bone by use of avidin-biotin-immunoperoxidase and immunogold-silver staining procedures. Low melting-point, paraffin-embedded sections of periodate lysine-paraformaldehyde-fixed undecalcified bone were used for immunostaining. We suggest that the combination of intact embryonic bone with this fixative and the immunohistochemical procedures used in this study may have helped to preserve antigenicity and thus to improve the efficiency of immunolabeling. Similar patterns of alkaline phosphatase localization were produced by the immunoperoxidase and immunogold-silver staining methods. The latter, although free of immunoreagents such as diaminobenzidine, must be monitored closely to avoid nonspecific staining during the silver enhancement procedure. Both methods revealed a concentration of the enzyme in osteoblasts and in areas of osteoid that lined the bone trabeculae. The results support the findings of earlier enzyme cytochemical studies in which osteoblasts were shown to have significant alkaline phosphatase activity.     

Calcium in Cells of Fresh Bone Stained with Glyoxal Bis(2-Hydroxyanil)

Fresh ileum of adult rats and vertebrae and calvariae of newborn rats were immersed in a staining solution containing 0.1 gm of glyoxal bis(2-hydroxyanil) (GBHA) per 2 ml of 3.4% NaOH in 75% ethanol, dehydrated in absolute ethanol, cleared in xylene, and embedded in paraffin. Paraffin sections of stained material, 7 μ thick, were affixed to albumenized slides, immersed in 90% ethanol saturated with Na₂CO₄ and KCN to ensure specificity for calcium, rinsed in 95% ethanol, counterstained in 50% ethanol containing 0.1% methylene blue, dehydrated in absolute ethanol, deparaffinized and cleared in xylene, and mounted in neutral synthetic resin. By this procedure, red Ca-GBHA granules were deposited in goblet and Paneth cells, and in the cytoplasm of osteoblasts, osteocytes, chondrocytes, and periosteal cells of developing bones. Calcium in apatite did not stain. In osseous tissues sectioned in a cryostat or processed by the freeze-dry or freeze-substitution method, epiphyseal chondrocyte calcium was removed, and apatite stained so intensely red that it obscured calcium in the bone cells. Failure of control osseous tissues to stain after immersion in a 1% solution of disodium salt of ethylenediaminetetraacetic acid or in the alcoholic, alkaline solvent of the GBHA solution, indicated that the red granules in the cells of developing bone were due to calcium present in the cells in vivo and not due to absorption of GBHA by tissue components other than calcium, or to diffusion of Ca⁺⁺ during staining. Calcium localized in the cytoplasm and processes of the osteogenic cells suggests the need to re-evaluate the role of osteoblasts as depositors of calcium during osteogenesis.     

Conversion of gastric mucosa to intestinal metaplasia in Cdx2-expressing transgenic mice

Gastric intestinal metaplasia occurs as a pathological condition in the gastric mucosa. To clarify how an intestine-specific homeobox gene, Cdx2, affects the morphogenesis of gastric mucosa, we generated transgenic mice expressing Cdx2 in parietal cells. Until Day 18 after birth, the number of parietal cells inthegastric mucosa of transgenic mice was the same as for their normal littermates. However, at Day 19, we detected several glands in which parietal cells disappeared and the proliferating zone moved from the isthmus to the base of the glands. Thereafter, parietal cells decreased gradually and disappeared at Day 37. All of the gastric mucosal cells, except for enterochromaffin-like (ECL) cells, were completely replaced by intestinal metaplasia, consisting of goblet cells, enteroendocrine cells, and absorptive cells expressing alkaline phosphatase. Pseudopyloric gland metaplasia was also formed. The transgenic mouse is a very useful model for clarifying physiological differentiation of gastric and intestinal cell lineages and analyzing the molecular events from intestinal metaplasia to adenocarcinoma.