Immunohistochemical demonstration of glycoconjugates bearing the type 2 chain-backbone structure in human fetal, normal and neoplastic gastrointestinal tract.

Immunohistochemical distributions of carbohydrate antigens based on the type 2 chain in normal as well as fetal and neoplastic tissues of human gastrointestinal tract were investigated with a monoclonal antibody (MAb) H11 (specific for type 2 chain) alone and in combination with the two MAbs MSG15 (for alpha 2----6 sialylated type 2 chain) and IB9 (for the alpha 2----6 sialylated type 2 chain and glycoproteins having NeuAc alpha 2----6Gal-NAc), and 188C1 (for short- and long-chain Lex antigens) and FH2 (for the long-chain Lex antigen). In the pyloric mucosa of secretors, the type 2 chain is oncodevelopmentally expressed, but in non-secretors it is detected in surface mucous cells of normal gastric mucosa. The alpha 2----6 sialylation, which is confined to endocrine cells of normal pyloric mucosa, occurs in fetal and carcinoma tissues. Irrespective of the secretor status, the short- and the long-chain Lex antigens can be detected in mature and immature glandular mucous cells of normal gastric mucosa, respectively; both antigens are also expressed in fetal and carcinoma tissues. In the colon, the type 2 chain and its alpha 2----6 sialylated counterpart are expressed in an oncodevelopmental manner. The short- and the long-chain Lex antigens are significantly enhanced in colonic carcinoma. The glycoproteins with NeuAc alpha 2----6GalNAc residues appear in gastric and colonic carcinoma as well as intestinalized gastric mucosa and transitional mucosa. Thus, some of these antigens were distinctively expressed in certain epithelial cells lining the normal gastrointestinal tract depending on maturation and patients' secretor status, and some were oncodevelopmental or carcinoma-associated antigens of the human gastrointestinal tract.     

Immunohistochemical demonstration of GABAB receptors in the rat gastrointestinal tract

Immunohistochemical localization of GABA_B-receptors was demonstrated in the rat gastrointestinal tract using a monoclonal antibody (GB-1) raised against the purified GABA_B-receptor. Immunoreactive staining for GABA_B-receptors was found in some populations of endocrine, muscular and neuronal components in the stomach and gut wall. Positive mucosal epithelial, probably endocrine, cells were distributed throughout the stomach and intestine. Double immunostaining indicated that such positive cells for GABA_B-receptors often co-possessed serotonin in the small intestine but not in the gastric body. In the muscular layer of the digestive canal, positive staining was seen as dotty granules punctuated on the surface of muscle fibers. In the enteric nervous system, positive neuronal somata were found in both submucosal and myenteric ganglia throught the entire canal extending from the stomach to the rectum. This is the first report to visualize the cellular localization of GABA_B-receptors in the gastrointestinal system of the rat, and should provide a fundamental basis for future studies on gastrointestinal functions regulated by GABA_B-receptors. Special issue dedicated to Dr. Kinya Kuriyama.     

Immunohistochemical demonstration of EC cells in rat gastrointestinal tract

Summary Selective immunohistochemical demonstration of enterochromaffin (EC) cells in the rat gastrointestinal tract has been carried out, using a highly specific rabbit antiserotonin (5-HT) serum and the unlabeled peroxidase-antiperoxidase complex (PAP), technique. The daily profile of the EC cell density was also studied using this method. No significant change was recorded.     

Immunohistochemical localization of BAX and BAD in the normal and BCL-2 null gastrointestinal tract

BAX and BAD are members of the BCL-2 family of proteins. The over-expression of BAX protein has been shown to accelerate apoptosis and increasedbax mRNA levels have also been shown to be associated with the initiation of apoptosis. BAD has also been shown to accelerate apoptosis. In this paper we describe the localization of BAD and BAX expression throughout the gastrointestinal tract of the mouse and the effect that BCL-2 has on the expression of these two proteins. We have discussed the distribution of BAX and BAD in relation to the differences between the small and large bowel in (i) the susceptibility of stem cells to apoptosis and (ii) tumour incidence.This work was supported by the Cancer Research Campaign.     

Immunohistochemical analysis of human neuronectin expression in normal, reactive, and neoplastic tissues

Neuronectin (NEC1) is a human extracellular matrix protein of central nervous system (CNS) parenchyma found throughout the white matter of rostral brain segments (telencephalon, diencephalon, some areas of mesencephalon), but not in rostral CNS gray matter, most areas of mesencephalon, pons, cerebellum, medulla, spinal cord, or peripheral nerves. The present immunohistochemical study, using two monoclonal antibodies to distinct epitopes on the NEC1 molecule, examined NEC1 expression in normal non-neural tissues, malignant tumors of diverse histological types, non-malignant skin lesions, and dermal incision wounds. We show that (a) NEC1 is expressed in normal fetal precartilage blastemas and fetal and adult vascular and visceral smooth muscle, but not in most loose connective tissues and skeletal or cardiac muscle; (b) NEC1 is found along epithelial-mesenchymal junctions, with marked differences in prevalence and histological patterns in different organs; and (c) mesenchymal activation associated with wound healing, actinic keratosis, psoriasis, and neoplasia leads to strong induction of NEC1 expression. Parallel studies with cultured human cells suggest that region-specific NEC1 expression in normal developing tissues and localized induction in wound healing and disparate diseases is under the control of extrinsic signals provided by regulatory polypeptides.     

Immunohistochemical localization of heparan sulfate proteoglycan in human gastrointestinal tract

Heparan sulfate proteoglycans (HS-PGs) are associated with important cell functions, for example, cell motility, cell adhesion, and oncogenesis. We examined the localization of HS-PGs in normal and carcinoma tissues of the gastrointestinal tract to help elucidate their roles in these organs. Fresh surgical materials from 134 patients with carcinoma of the stomach or large intestine and 26 patients with various diseases of the small intestine were immunostained after fixation with 10E4 (an antibody against the HS of HS-PG) as a primary antibody. Immunoelectron microscopy (immunogold method) was also performed. The basolateral surfaces of normal tissues of the large and small intestines were strongly stained with antibody confirmed by electron microscopy. In the stomach, lesions with intestinal metaplasia showed the same staining as the intestines, although normal gastric tissue showed staining only in some parts of the basal layer of fundic and pyloric glands. Carcinoma tissues in all cases examined showed staining with antibody. Better results were obtained after fixation in acetic alcohol or zinc-containing solutions than in ordinary formalin. These characteristic localizations of HS-PG in intestines and stomachs suggest that this kind of HS-PG staining could be a hallmark characteristic of the intestine.     

Immunohistochemical demonstration of villin in the normal human pancreas and in chronic pancreatitis

Summary Human pancreatic tissue was investigated by immunohistochemistry using a polyclonal antibody against the actin binding protein villin, which participates in the formation of actin filament bundles in the microvilli. In cells of the different parts of the pancreatic duct system as well as in the acinar cells villin immunoreactivity was located mainly at the apical cell surface. This was confirmed by the ultrastructural demonstration of microvilli on the surface of duct and acinar cells, which exhibited the typical actin bundles. In chronic pancreatitis the staining for villin in duct-like structures of degenerative pancreatic tissue was irregular or even absent. This correlated with the electron microscopic observation of duct-like structures known as tubular complexes composed of cells devoid of microvilli at the apical cell surface. At the light microscopical level degenerative structures without lumen and of unknown origin showed a strong staining for villin at their basal cell surface.     

Immunohistochemical demonstration of villin in the normal human pancreas and in chronic pancreatitis

Human pancreatic tissue was investigated by immunohistochemistry using a polyclonal antibody against the actin binding protein villin, which participates in the formation of actin filament bundles in the microvilli. In cells of the different parts of the pancreatic duct system as well as in the acinar cells villin immunoreactivity was located mainly at the apical cell surface. This was confirmed by the ultrastructural demonstration of microvilli on the surface of duct and acinar cells, which exhibited the typical actin bundles. In chronic pancreatitis the staining for villin in duct-like structures of degenerative pancreatic tissue was irregular or even absent. This correlated with the electron microscopic observation of duct-like structures known as tubular complexes composed of cells devoid of microvilli at the apical cell surface. At the light microscopical level degenerative structures without lumen and of unknown origin showed a strong staining for villin at their basal cell surface.     

Immunohistochemical demonstration of somatostatin in the pancreas of fetal and adult sheep

Pancreatic cells immunoreactive with rabbit anti-somatostatin serum were demonstrated in fetal and adult sheep by immunofluorescence histochemistry. Our observations provide evidence that in the sheep the pancreas is a major site of production of the hormone. It is suggested that the hormone may be important in modulating intra- and extra-islet function.     

Protein phosphorylation substrates in normal and neoplastic squamous epithelia of the human upper aero-digestive tract

Protein phosphorylation was studied in crude and in protein kinase C (Pk-C)-enriched preparations from squamous cell carcinomas and normal mucosa of the human upper aero-digestive tract. In crude soluble preparations from neoplastic mucosa we found a 5-fold higher basal endogenous phosphorylation when compared to normal mucosa. In particulate fractions the increase was 3-fold. SDS-PAGE and autoradiography of phosphorylated proteins in crude soluble tumor extracts showed bands corresponding to proteins with apparent molecular weights of 18, 37, 40-42, 52, 60, 62 and 90 kDa. In normal mucosa the phosphorylation of these proteins was very low or absent, except for the proteins with molecular weights of 40-42 and 52-55 kDa. Addition of Ca2+ or Ca2+/phospholipids to the reaction mixture caused phosphorylation of additional proteins with apparent molecular weight of 45-50 kDa in soluble preparations of tumors. Cyclic AMP or cGMP had no significant effect on the phosphorylation of endogenous proteins. In the partially purified, Pk-C-enriched fractions the phosphorylation in the presence of Ca2+/phospholipids was distinctly higher in tumors when compared to the phosphorylation observed in normal mucosa, and some phosphorylation substrates were detected only in tumor tissue. In order to find out whether the elevated basal phosphorylation was due to an endogenous activation of protein kinases, different inhibitors of serine/threonine protein kinases were tested. These inhibitors included: heat-stable cyclic AMP-dependent protein kinase (Pk-A) inhibitor, Pk-A inhibitor peptide (Wiptide), heparin and the Pk-C inhibitors peptide 19-36 and H-7. None of these inhibitors had any significant effect on the basal phosphorylation. In conclusion, our results show the existence of endogenous phosphorylation substrates in human squamous cell carcinomas from the upper aerodigestive tract, and indicates that there is a significantly higher basal and Pk-C specific phosphorylation of endogenous substrates in tumors compared to normal mucosa. This may be of importance for the transformation and altered growth regulation in epithelial tumors.     

Immunohistochemical demonstration of proteinase inhibitor alpha-1-antichymotrypsin in normal human central nervous system

The presence of alpha-1-antichymotrypsin, a serine proteinase inhibitor with a high affinity for cathepsin G, is demonstrated in the normal human central nervous system (CNS) by immunohistochemical techniques. Paraffin-embedded normal human CNS tissue from five adult, two fetal, one neonatal and three newborn autopsies were stained with monospecific rabbit antibodies to human alpha-1-antichymotrypsin using biotinylated goat anti-rabbit antibodies and an avidinbiotin-peroxidase complex. Positive immunostaining was seen in neurons and glial cells in the cerebral cortex, basal ganglia, hippocampus, cerebellum, brainstem, and spinal cord of the adults. The epithelium of the adult choroid plexus had the most intense staining in apical granular organelles corresponding in position to lysosomes or secretory granules. Ependymal cells, particularly those near the choroid plexus, were immunostained. The fetal CNS had no alpha-1-antichymotrypsin staining. Limited staining of choroid plexus, ependyma, and frontal lobe was found in the newborns. Immunostaining in the neonatal temporal lobe was only found in the choroid-plexus epithelium. These observations establish a widespread distribution of this proteinase inhibitor in the normal human CNS. Developmental regulation of this inhibitor in the human CNS is also indicated.     

Immunohistochemical study of the expression of fucosylated glycoconjugates in human lung carcinoma]

Lewis X, Sialyl Lewis X and Sialyl Dimeric Lewis X are three fucosylated glycoconjugates on cell surface. With immunohistochemical method, the expression of the three structures in the original lung cancer tissues (with or without metastasis), adjacent tissues and metastatic lesions of lung carcinoma were studied. It was found that the three antigens were expressed with different intensity on the cell surface and in the cytoplasm of the lung carcinoma cell. However, there was no or only trace expression of these antigens in the adjacent tissues of lung carcinoma and normal lung tissues. Moreover, the original lesions of lung carcinoma with metastasis and/or poor differentiation expressed higher level of the three antigens than those without metastasis and/or with well or medium differentiation. Sialyl Lewis X was considered to be more closely related to the metastatic ability and differentiation of the lung carcinoma cell than the other two antigens, Lewis X and Sialyl Dimeric Lewis X. Furthermore, in the lymph nodes with lung carcinoma cell metastasis, there were expression of the three antigens with different degree, while in those lymph nodes without lung carcinoma cell metastasis, there was no expression of the three antigens.     

Immunohistochemical localization of Zn-alpha 2-glycoprotein in normal human tissues

The Zn-alpha 2-glycoprotein (Zn-alpha 2-GP) is present at a high concentration in the seminal plasma and at significant levels in other human body fluids. Its precise localization, however, has remained unclear, as well as its physiological and pathological significance. The present study reports the immunohistochemical localization of this protein in normal adult human tissues. Localization of the reactive product to anti-human plasma Zn-alpha 2-GP antibody was demonstrated in the following cells: luminal and basal cells of the prostate gland, luminal epithelial cells of the acini and of some ducts of the mammary glands, luminal cells of the secretory portion of the eccrine and apocrine sweat glands, serous cells of the salivary, tracheal, and bronchial glands, acinar cells of the esophageal glands, exocrine acinar cells of the pancreas, hepatocytes of the liver, and epithelial cells of the proximal and distal tubules in the kidney. The present results suggest that Zn-alpha 2-GP exerts some unknown but fairly widespread exocrine function and may be produced in the various epithelial cells tested. Hepatocytes are also suggested to be a source of the protein in the blood plasma.     

Immunohistochemical demonstration of alpha1,4-N-acetylglucosaminyltransferase that forms GlcNAcalpha1,4Galbeta residues in human gastrointestinal mucosa.

alpha1,4-N-acetylglucosaminyltransferase (alpha4GnT) is a glycosyltransferase that mediates transfer of GlcNAc to betaGal residues with alpha1,4-linkage, forming GlcNAcalpha1--> 4Galbeta-->R structures. In normal human tissues, glycoproteins having GlcNAcalpha1-->4Galbeta-->R structures at non-reducing terminals are exclusively limited to the mucins secreted from glandular mucous cells of gastric mucosa, Brunner's gland of duodenum, and accessory gland of pancreaticobiliary tract. Recently, we have isolated a cDNA encoding human alpha4GnT by expression cloning. Although alpha4GnT plays a key role in producing this unique glycan in vitro, the actual localization of alpha4GnT was not determined. In this study we examined the localization of alpha4GnT in various human tissues, including gastrointestinal mucosa, using a newly developed antibody against human alpha4GnT. The specificity of the antibody was confirmed by analyses of human gastric adenocarcinoma AGS cells transfected by alpha4GnT cDNA. Expression of alpha4GnT was largely associated with the Golgi region of mucous cells that produce the mucous glycoproteins having GlcNAcalpha1-->4Galbeta-->R, such as the glandular mucous cells of stomach and Brunner's gland. An immunoprecipitation experiment disclosed that two distinct mucin proteins, MUC5AC and MUC6 present in gastric mucin, carried the GlcNAcalpha1-->4Galbeta-->R structures. These results indicate that alpha4GnT is critical to form the mucous glycoproteins having GlcNAcalpha1-->4Galbeta-->R on MUC6 and MUC5AC in vivo.(J Histochem Cytochem 49:587-596, 2001)     

Immunohistochemical demonstration of a new thiamine diphosphate-binding protein in the rat digestive tract

Summary We purified a new thiamine diphosphate-binding protein (ThDP-BP), produced an antiserum against it, and examined its immunohistochemical distribution in the rat gastrointestinal tract using the avidinbiotin complex method. Positive staining for ThDP-BP was found in the epithelial glands of the stomach, small intestine and large intestine, and in the nuclei of hepatocytes. Measurement of the content of thiamine and its phosphate esters in extracts from the gastric and intestinal mucosa also indicated the presence of ThDP-BP in the stomach and intestinal mucosa. ThDP-BP may be useful for investigating the absorption and metabolism of the thiamine in metabolic disorders.     

Immunohistochemical localization of sex hormone-binding globulin in normal and neoplastic breast tissue.

The distribution of sex hormone-binding globulin-like antigens (SHBG-LA) in normal and neoplastic human breast tissues was investigated by immunohistochemistry, employing a monospecific polyclonal antiserum against highly purified human SHBG and an avidin-biotin-peroxidase complex (ABC) method in formalin-fixed paraffin embedded tissue sections. In normal breast tissues the staining of SHBG-LA was present exclusively in the cytoplasm of epithelial cells of ductal and ductular types. Nuclei as well as stromal and lymphatic tissues remained unstained. While the staining was positive in all cases of intraductal carcinoma, only 4 out of 15 infiltrating carcinomas revealed SHBG-LA. The demonstration of a plasma sex steroid binding globulin in the cytoplasm of endocrine target cells is consistent with the hypothesis that steroid-binding globulins are able to enter target cells. The apparent loss of this specific cell function in infiltrating carcinomas may result from dedifferentiation and change of cell membrane properties occurring during the process of neoplastic progression.     

Gangliosides of normal and neoplastic human melanocytes

The major ganglioside component isolated from diploid human melanocytes is sialosyllactosylceramide (GM3 86-91% of total sialic acid). The corresponding disialo derivative (GD3) is found as a minor component (2-6% of total sialic acid) in the membranes of these cells. In human melanoma cells, grown in tissue culture, GD3 is the predominant ganglioside component (48-63% of total sialic acid). Withdrawal of TPA from the culture medium of normal melanocytes or addition of TPA to the medium of melanoma cells had no significant effect on GM3/GD3 ratios. We conclude that the difference between the composition of gangliosides is related to the normal vs transformed phenotypes of melanocytes.     

Synthesis of plasma proteins in fetal, adult, and neoplastic human brain tissue

The synthesis of plasma proteins directed by mRNA from human brain tissues was studied by combining in vitro or in ovo translation of mRNAs with crossed immunoelectrophoresis of the mRNA-directed labeled polypeptides, followed by autoradiography of the washed plates. Poly(A)-containing mRNA was prepared from different developmental stages of fetal and postnatal human brain and also from primary glioblastomas and meningiomas. Several plasma protein-like polypeptides were identified in the autoradiographs by their migration coordinates in the two-dimensional gels, compared with immunoprecipitates formed by mature, unlabeled, stainable proteins. These included polypeptides migrating like Gc globulin, haptoglobin, fibrinogen, alpha-fetoprotein, transferrin, cholinesterase, and alpha 2-macroglobulin; other, yet unidentified plasma proteins, were also observed. In general, the synthesis of these plasma proteins appeared to be more pronounced in fetal and neoplastic brain tissues than in postnatal tissues. However, clear immunoprecipitates for some of these plasma proteins could also be detected in products directed by mRNA from particular regions of mature, normal brains, indicating that some synthesis of plasma proteins takes place in the human brain even as late as 40 years of age. mRNAs for several proteins were also identified in samples of neoplastic brain. mRNA for transferrin was identified in normal fetal and adult brain but not in either the glioblastomas or meningiomas studied. Microinjected Xenopus oocytes, in which post-translational processing occurs as well, were also used to translate fetal brain mRNA. Several plasma proteins could be detected in the translation products which were induced and stored in the oocytes. These included hemopexin, which could not be detected in the in vitro system. Others, such as cholinesterase, were found to be secreted by the oocytes. These findings indicate that different cell types in the human brain may produce and either store or secrete particular plasma proteins at defined stages in their development.     

人类正常胃肠道组织基因表达谱特征的生物信息学分析

目的:筛选人类正常胃肠道组织特异性高表达基因.方法:以公开发表的人类正常组织基因表达数据库资料为研究对象,应用单侧 Student T检验筛选胃、回肠和结肠组织的特异性高表达基因.Ingenuity软件和KEGG分析特异性高表达基因相关的生理功能;Cluster软件分析胃特异性高表达基因谱在胃癌基因数据库中的表达特征.结果:筛选出胃高表达基因196个,回肠高表达基因203个,结肠高表达基因224个,高表达基因的功能与胃肠道的主要生理功能相吻合.筛选出的正常胃肠高表达基因中含有一些癌基因和抑癌基因.系统聚类分析发现胃特异性高表达基因在胃癌数据库的胃正常组织中高表达,而相应的胃癌组织低表达,癌组织和正常组织可以完好聚类分组,并且可以区分中分化和低分化胃癌.结论:人类正常胃、回肠和结肠具有组织特异的与生理功能密切相关的高表达基因群.     

Microbial biofilms in the human gastrointestinal tract

The human gastrointestinal tract contains rich and diverse microbiotas along its length. However, while extensive studies have been made on lumenal bacterial communities in the gut, less work has been carried out on organisms growing in biofilms, where individual groups of bacteria exist in a multiplicity of different microhabitats and metabolic niches associated with the mucosa, the mucus layer and particulate surfaces in the gut lumen. Bacteria and yeasts also occur in biofilms attached to artificial surfaces and devices implanted in the host, such as in patients being fed via enteral tubes. Although we are just beginning to investigate the composition and metabolic activities of these structures, increasing evidence suggests that they are important to the host in both health and disease. There is mounting interest in mucosal biofilms in the colon, especially with respect to their role in inflammatory bowel disease. Because bacteria growing in biofilms are more resistant to antibiotics than unattached organisms, it is often difficult to modify the structure and composition of these communities, or to eradicate them from the body. However, recent work has shown that there is considerable potential to alter the species composition of mucosal biofilms in a beneficial way using synbiotics.