Mucin expression in pleomorphic adenoma of salivary gland: a potential role for MUC1 as a marker to predict recurrence.

BACKGROUND: Pleomorphic adenoma of the salivary gland (PA) is essentially a benign neoplasm. However, patients with recurrent PA are difficult to manage. There are rare reports on useful immunohistochemical markers to detect a high risk of recurrence when the primary lesions are resected. AIMS: To find a new marker to predict the recurrence of PA. METHODS: Primary lesions of PA were collected from nine patients showing subsequent recurrence and from 40 patients without recurrence during at least 10 years of follow up of the disease. Paraffin wax embedded tumour samples of the two groups were examined for the expression profiles of MUC1 (differentially glycosylated forms), MUC2, MUC4, MUC5AC, and MUC6 using immunohistochemistry. Several clinicopathological factors were also examined. RESULTS: In univariate analysis of the factors examined, MUC1/DF3 high expression (more than 30% of the neoplastic cells stained) in the primary lesions was seen more frequently in patients with recurrence (four of nine) than in those without recurrence (three of 40; p = 0.011). Larger tumour size (more than 3.0 cm) of the primary PA was also a significant (p = 0.035) risk factor for the recurrence of PA. In multivariate analysis, only high expression of MUC1/DF3 was found to be a significant independent risk factor for the recurrence of PA (p = 0.021). CONCLUSIONS: Expression of MUC1/DF3 in PA is a useful marker to predict its recurrence. Those patients with PA showing positive MUC1/DF3 expression should be followed up carefully.

Model for paraaortic lymph node metastasis produced by orthotopic implantation of ovarian carcinoma cells in athymic nude mice.

Lymph node metastasis through the lymphatic vessels is a critical step in determining the outcome of ovarian cancer patients, and prognosis should be improved by preventing lymph node metastasis. However, experimental models for lymph node metastasis of ovarian carcinoma are not available. We developed an orthotopic transplantation model to study this process in nude mice using the human ovarian carcinoma cell lines, KF and MH. Highly metastatic sublines (KF-LN3 and MH-LN3) were selected in vivo in nude mice by repeated orthotopic transplantation, lymph node metastasis formation and culturing the tumour cells in vitro. Because this model seems to correspond to the advanced clinical stage of ovarian carcinomas, it should be useful in understanding the molecular biology of ovarian carcinomas and in the development of therapeutic modalities against lymph node metastasis.

Expression of mucins (MUC1, MUC2, MUC5AC and MUC6) in mucinous carcinoma of the breast: comparison with invasive ductal carcinoma.

AIMS: Mucinous carcinoma of the breast usually shows less frequent lymph node metastasis and more favourable outcome compared with invasive ductal carcinoma. The aim of this study is to compare the expression profiles of several mucins in mucinous carcinomas and invasive ductal carcinomas to gain insight into the relationship between the less aggressive biological nature of mucinous carcinoma and the role of mucins. METHODS AND RESULTS: We examined the expression profiles of MUC1 (membrane-bound mucin) of different glycoforms (from non-glycosylated form to fully glycosylated form), MUC2 (intestinal type secretory mucin), MUC5AC (gastric surface type secretory mucin) and MUC6 (gastric pyloric gland type secretory mucin) in 17 mucinous carcinomas and 46 invasive ductal carcinomas using immunohistochemistry. Various glycoforms of MUC1 were expressed frequently in both mucinous carcinomas (65-100%) and invasive ductal carcinomas (92-100%), although non-glycosylated MUC1 (MUC1/CORE) and fully glycosylated MUC1 (MUC1/HMFG-1) showed significantly lower expression rates in mucinous carcinomas compared with those in invasive ductal carcinomas. The expression rates of MUC2 (94%) and MUC6 (71%) in mucinous carcinomas were significantly higher than those of MUC2 (15%) and MUC6 (15%) in invasive ductal carcinomas. There was no significant difference in the expression rate of MUC5AC in mucinous carcinomas (12%) and that in invasive ductal carcinomas (4%). CONCLUSIONS: The expression rate of MUC1/CORE and MUC1/HMFG-1, which is related to poor prognosis in the gastric and colorectal cancers, is low in mucinous carcinomas. The high expression rate of gel-forming secretory mucins (MUC2 and MUC6) in mucinous carcinoma suggests that high production of these types of mucins may act as a barrier to cancerous extension resulting in their less aggressive biological behaviour.

Kupffer cell-mediated recruitment of rat dendritic cells to the liver: roles of N-acetylgalactosamine-specific sugar receptors.

BACKGROUND & AIMS: Tissue recruitment of dendritic cells (DCs) is essential for antigen presentation. This study aimed to examine cellular and molecular mechanisms for DC recruitment to the liver. METHODS: Purified rat DCs were injected into circulation and their traffics were analyzed in normal and Kupffer cell-depleted rats by intravital confocal microscopy and immunohistology. Affinities of DCs to sinusoidal cells were examined by a cell-binding assay. DC precursor recruitment was induced by particulate injection. RESULTS: Both DC precursors and DCs at the antigen-transporting stage could be recruited to the liver, and their majority initially showed a selective binding to Kupffer cells. In the Kupffer cell-depleted rats, DCs could neither be recruited to the liver nor adhere to sinusoidal walls. Pretreatment with varied monosaccharides showed that sugar residues consisting of N-acetylgalactosamine were necessary for this binding. The binding was calcium-dependent, implying the C-type lectin involvement. Furthermore, DCs could endocytose N-acetylgalactosamine polymers in a receptor-specific manner. CONCLUSIONS: The DC-Kupffer cell binding through N-acetylgalactosamine-specific C-type lectin-like receptors is crucial for DC recruitment to the liver. Rat DCs at least partly possess receptors for endocytosis of galactosylated antigens. These DC receptors as well as Kupffer cell lectins are presumably responsible for this binding.

N-acetylgalactosamine incorporation into a peptide containing consecutive threonine residues by UDP-N-acetyl-D-galactosaminide:polypeptide N-acetylgalactosaminyltransferases.

A limited number of glycosylation products were generated in a cell-free system from a portion of the MUC2 tandem repeat, PTTTPITTTTK, when microsome fractions of human colon carcinoma LS174T cells were used as the source of UDP-N-acetyl-D-galactosaminide:polypeptide N-acetylgalactosaminyltransferases (pp-GalNAc-T) in our previous work. The structures of all products suggested that there were only two biosynthetic pathways in the GalNAc incorporation into this peptide. In the present report, the putative biosynthetic intermediates, PTTT*PITTTTK (asterisk designates a GalNAc residue), PT*TTPITTTTK, PTT*T*PITT*T*TK, and PT*TTPIT*T*T*TK, of these two hypothetical pathways were used as acceptors to prove that these two pathways do exist. The incubation products of these glycopeptides, microsome fractions of LS174T cells, and UDP-GalNAc were fractionated by reverse-phase HPLC and their structures were determined using MALDI-TOF MS and peptide sequencing. The products from PTTT*PITTTTK were PTTT*PITTT*TK, PTTT*PITT*T*TK, PTT*T*PI-TT*T*TK, PTT*T*PIT*T*T*TK, PT*T*T*PIT*T*T*TK, and PT*T*T*PIT*T*T*T*K. The products from PTT*-T*PITT*T*TK exactly corresponded to the products with five to seven GalNAc residues from PTTT*PITTTTK. The products from PT*TTPITTTTK were PT*TTPITT*TTK, PT*TTPIT*T*TTK, and PT*TTPIT*T*T*TK. PT*TTP-IT*T*T*TK was not converted further under the applied condition. All the products detected and analyzed were the same as those obtained when the unsubstituted peptide and microsome fractions of LS174T cells were incubated. Immunocytochemical analysis indicated that LS174T cells contain at least four pp-GalNAc-Ts (-T1, -T2, -T3, and -T4), suggesting that control of the order and the maximum number of GalNAc incorporation into this peptide is regulated through the coordinated actions of these and possibly other pp-GalNAc-Ts.

Distinct orders of GalNAc incorporation into a peptide with consecutive threonines.

Mucin O-glycosylation is initiated by a transfer of N-acetyl-d-galactosamine (GalNAc) to Ser and Thr residues in polypeptides with a family of UDP-GalNAc:polypeptide N-acetylgalactosaminyltransferases (pp-GalNAc-Ts). In this paper, four human pp-GalNAc-Ts (pp-GalNAc-T1, T2, T3, and T4) were tested for their preferential orders of GalNAc incorporation into FITC-PTTTPITTTTK, a portion of the tandem repeat of human MUC2. The products were separated by reverse-phase HPLC and characterized by MALDI-TOF MS and peptide sequencing. pp-GalNAc-T1 showed preference for acceptor sites, but the order of the incorporation into these sites seemed to be random. In contrast, the GalNAc incorporation by pp-GalNAc-T2, T3, or T4 was not only site-specific but also according to the specific orders. Furthermore, pp-GalNAc-T2, T3, or T4 had distinct maximum numbers of GalNAc incorporations into this peptide.

A remodeling system of the 3'-sulfo-Lewis a and 3'-sulfo-Lewis x epitopes.

It has been reported that the chemically synthesized 3'-sulfo-Le(a) and 3'-sulfo-Le(x) epitopes have a high potential as a ligand for selectins. To elucidate the physiological functions of 3'-sulfated Lewis epitopes, a remodeling system was developed using a combination of a betaGal-3-O-sulfotransferase GP3ST, hitherto known alpha1,3/1,4-fucosyltransferases (FucT-III, IV, V, VI, VII, and IX) and arylsulfatase A. The pyridylaminated (PA) lacto-N-tetraose (Galbeta1-3GlcNAcbeta1-3Galbeta1-4Glc) was first converted to 3'-sulfolacto-N-fucopentaose II (sulfo-3Galbeta1-3(Fucalpha1-4)GlcNAcbeta1-3Galbeta1-4Glc)-PA by sequential reactions with GP3ST and FucT-III. The 3'-sulfolacto-N-fucopentaose III (sulfo-3Galbeta1-4(Fucalpha1-3)GlcNAcbeta1-3Galbeta1-4Glc)-PA was then synthesized from lacto-N-neotetraose (Galbeta1-4GlcNAcbeta1-3Galbeta1-4Glc)-PA by GP3ST and FucT-III, -IV, -V, -VI, -VII, or -IX in a similar manner. The substrate specificity for the 3'-sulfated acceptor of the alpha1,3-fucosyltransferases was considerably different from that for the non-substituted and 3'-sialylated varieties. When the GP3ST gene was introduced into A549 and Chinese hamster ovary cells expressing FucT-III, they began to express 3'-sulfo-Le(a) and 3'-sulfo-Le(x) epitopes, respectively, suggesting that GP3ST is responsible for their biosynthesis in vivo. The expression of the 3'-sialyl-Le(x) epitope on Chinese hamster ovary cells was attenuated by the introduction of GP3ST gene, indicating that GP3ST and alpha2,3-sialyltransferase compete for the common Galbeta1-4GlcNAc-R oligosaccharides. Last, arylsulfatase A, which is a lysosomal hydrolase that catalyzes the desulfation of 3-O-sulfogalactosyl residues in glycolipids, was found to hydrolyze the sulfate ester bond on the 3'-sulfo-Le(x) (type 2 chain) but not that on the 3'-sulfo-Le(a) (type 1 chain). The present remodeling system might be of potential use as a tool for the study of the physiological roles of 3'-sulfated Lewis epitopes, including interaction with selectins.

Intestinal epithelial cells express and secrete the CD43 glycoform that contains core 2 O-glycans.

Several glycoforms of CD43 are known to regulate cellular interactions in the immune system. One such glycoform, the CD43 that bears core 2 O-glycans, is also known to be expressed on T lymphocytes and natural killer cells, but only after their activation. Previous studies have also shown that when Caco-2 cells, which are derived from human colon carcinoma, differentiate into enterocytes, they also express core 2 O-glycans, though proteins bearing this glycan are unknown. To examine whether CD43 glycosylation is altered during enterocytic differentiation of Caco-2 cells, we conducted immunocytochemical studies with a monoclonal antibody, 1D4, that recognizes a glycoform of CD43 bearing core 2 O-glycans. We found that 1D4 could bind to intracellular granules but not the cell surface of differentiated Caco-2 cells, whereas hematopoietic cells expressed 1D4 epitope on the cell surface as previously shown. The reactivity with this antibody increased as the degree of cell differentiation progressed as shown by the activity of the apical enzyme marker, dipeptidyl peptidase IV. 1D4-reactive CD43 was also found in the culture medium of differentiated Caco-2 cells, suggesting this molecule may be stored and secreted. The production and secretion of this CD43 glycoform by enterocyte-like Caco-2 cells was enhanced, and most 1D4 epitope converted to a soluble form when bacterial lipopolysaccharide was present. These observations strongly support the possibility that core 2 O-glycans on mucins such as CD43 are important to primary defense on the intestinal epithelium against infection.

Expression of MUC1 mucins in the subserosal layer correlates with postsurgical prognosis of pathological tumor stage 2 carcinoma of the gallbladder.

The overall outcome of pT(2) gallbladder carcinoma has not been favorable. Postsurgical recurrence at distant sites occurs in some cases, although the carcinoma was limited to the gallbladder wall. A high level expression of MUC1 mucins with sialylated carbohydrates (sialylated MUC1 mucins) is correlated with poor survival in intrahepatic bile duct carcinoma. In the present study, immunohistochemistry was performed to determine the expression level of sialylated MUC1 mucins, detected by a monoclonal antibody, MY.1E12, in 31 cases of pT(2) gallbladder carcinoma on which curative resections had been performed and to determine the correlation of the expression level of MY.1E12-reactive-MUC1 mucin with mode of recurrence and postsurgical survival. Immunostainings of the MUC1 mucin were recognized in different types of noncancerous pathological epithelia of the gallbladder except for intestinal metaplasia and cancerous epithelia. Immunohistochemical localization was classified into apical, cytoplasmic, and stromal types based on the predominant cellular distribution of MY.1E12-reactive-MUC1 mucin. In 31 cases of pT(2) carcinoma, the localization was apical type in 64%, cytoplasmic type in 71%, and stromal type in 48% of the cases at the deepest invading sites in the subserosal layer. Distant recurrences, i.e., peritoneal dissemination in 8 patients and liver metastasis in 3 patients, were seen in 8 (53%) of 15 cases of pT(2) carcinoma that had > or =10% of the cancerous epithelia showing stromal localization of the MUC1 mucin at the deepest invading sites and in 2 (12%) of 16 cases that had <10% of those showing the stromal localization. The postsurgical survival outcome was significantly poorer in the former than in the latter (P = 0.044). In pT(2) gallbladder carcinoma, the presence of MY.1E12-reactive-MUC1 mucin in the stroma adjacent to the cancerous epithelia in the subserosal layer correlates with the aggressiveness of the disease, such as the tendency to form distant recurrences. This phenotype may serve as a unique biological feature associated with the malignant behavior of pT(2) gallbladder carcinoma.

Molecular cloning and characterization of UDP-GlcNAc:lactosylceramide beta 1,3-N-acetylglucosaminyltransferase (beta 3Gn-T5), an essential enzyme for the expression of HNK-1 and Lewis X epitopes on glycolipids.

A new member of the UDP-N-acetylglucosamine:beta-galactose beta1,3-N-acetylglucosaminyltransferase (beta3Gn-T) family having the beta3Gn-T motifs was cloned from rat and human cDNA libraries and named beta3Gn-T5 based on its position in a phylogenetic tree. We concluded that beta3Gn-T5 is the most feasible candidate for lactotriaosylceramide (Lc(3)Cer) synthase, an important enzyme which plays a key role in the synthesis of lacto- or neolacto-series carbohydrate chains on glycolipids. beta3Gn-T5 exhibited strong activity to transfer GlcNAc to glycolipid substrates, such as lactosylceramide (LacCer) and neolactotetraosylceramide (nLc(4)Cer; paragloboside), resulting in the synthesis of Lc(3)Cer and neolactopentaosylceramide (nLc(5)Cer), respectively. A marked decrease in LacCer and increase in nLc(4)Cer was detected in Namalwa cells stably expressing beta3Gn-T5. This indicated that beta3Gn-T5 exerted activity to synthesize Lc(3)Cer and decrease LacCer, followed by conversion to nLc(4)Cer via endogenous galactosylation. The following four findings further supported that beta3Gn-T5 is Lc(3)Cer synthase. 1) The beta3Gn-T5 transcript levels in various cells were consistent with the activity levels of Lc(3)Cer synthase in those cells. 2) The beta3Gn-T5 transcript was presented in various tissues and cultured cells. 3) The beta3Gn-T5 expression was up-regulated by stimulation with retinoic acid and down-regulated with 12-O-tetradecanoylphorbol-13-acetate in HL-60 cells. 4) The changes in beta3Gn-T5 transcript levels during the rat brain development were determined. Points 2, 3, and 4 were consistent with the Lc(3)Cer synthase activity reported previously.

Anti-metastatic effect of the sialyl Lewis-X analog GSC-150 on the human colon carcinoma derived cell line KM12-HX in the mouse.

We investigated the inhibitory effect of the sialyl Lewis-X (sLeX) analog, GSC-150, on hepatic metastasis of the human colon carcinoma derived cell line, KM12-HX, which highly expresses sLeX antigen on the cell surface. The number of cancer nodules found in BALB/c nude mouse liver 6 weeks after intrasplenic injection of KM12-HX cells was significantly reduced by co-administration of GSC-150. The amount of [3H]thymidine-labeled KM12-HX cells distributed in liver was also significantly reduced by GSC-150 co-administration in lipopolysaccharide (LPS)-treated mice at 48 h after administration of the tumor cells, while GSC-150 did not reduce the amount of HX cells distributed at 30 min. Considering our previous report that the initial phase of the distribution of KM12-HX cells in liver is governed by their being trapped in the hepatic microvessels because of their large size (Mizuno et al., J. Hepatol., 28, 865-877, 1998), these results suggest that GSC-150 does not inhibit this first-pass trapping by microvessels, but inhibits the subsequent process which is more directly related to final metastasis. GSC-150 inhibited the adhesion of KM12-HX cells to tumor necrosis factor-alpha (TNF-alpha)-activated human umbilical vein endothelial cells (HUVECs). These findings imply that the anti-metastatic effect of GSC-150 in vivo could be explained by its inhibition of cell-cell interactions between cancer and host cells.

Mutated plant lectin library useful to identify different cells.

The 24 nucleotides comprising the carbohydrate-recognition domain of Maackia amurensis hemagglutinin (MAH) cDNA were randomly mutated. The mutant lectins were expressed as glutathione-S-transferase fusion proteins in Escherichia coli and 16 clones were randomly chosen. Although all of 16 recombinant lectins reacted strongly with anti-MAH polyclonal antibody, the carbohydrate-recognition domain of each was unique. As shown by agglutination studies, each mutant MAH lectin was able to bind to erythrocytes from one or more of five animal species in very distinct patterns. Thus, novel plant lectin libraries can be used to discriminate in a highly specific manner among a variety of cell types. This technology may prove to be very useful in a number of different applications requiring a high level of specificity in cell identification.

Fibroblast migratory factor derived from mouse colon carcinoma cells: potential roles of fibronectin in tumor stroma formation.

Mouse colon carcinoma cell line colon 38 was used to investigate migratory factor for fibroblasts because marked fibrotic tissue was associated with these cells growing at transplanted sites and liver metastases in vivo. Migration-inducing activity was detected in the serum-free culture supernatants of colon 38 cells, as shown by the Boyden chamber assays using NIH3T3 cells as target cells. The active substance was partially purified by a combination of anion-exchange, hydrophobic, and gel-permeation chromatography. An approximate relative molecular mass of the active substance was estimated to be between 100,000 and 400,000, judging from the eluting position in the gel-permeation chromatography. The migratory activity in the partially purified preparation was removed by incubation with beads coated with an anti-mouse fibronectin antibody. NIH3T3 cells incubated in the presence of culture supernatants of colon 38 cells exhibited higher growth rate, organized actin filaments, and increased chondroitin sulfate and hyaluronan. Fibronectin did not elicit such effects and partially purified migratory factor showed relatively low activity in these regards. Thus, colon 38 cells seem to secrete fibronectin and other soluble substances, which induce tumor stroma formation through migration and activation of host fibroblasts.

Carbohydrate-mediated adhesion of human colon carcinoma cells to human liver sections.

Clinicopathological observations have revealed that the expression level of a carbohydrate antigen recognized by the monoclonal antibody (mAb) FH6 in colorectal carcinomas is higher at advanced stages than at early stages. The present study aimed to elucidate whether human colon carcinoma cell surface glycans recognized by mAb FH6 determine the ability of these cells to adhere to sections of human liver. Variant human colon carcinoma cell lines selected for high and low binding of mAb FH6 were compared with respect to their adhesive capacity. The cells expressing the higher level of FH6 binding also showed a greater ability to adhere to liver sections. This adhesion was not blocked by anti-E-selectin monoclonal antibodies, but pretreatment of the carcinoma cells with endo-beta-galactosidase significantly reduced both cell surface binding of mAb FH6 and the ability of the cells to adhere to liver sections. Our observations suggest that endo-beta-galactosidase-sensitive carbohydrate chains containing an epitope recognized by mAb FH6 play an important role in the adhesion of human colon carcinoma cells to human liver sections. Whether these interactions have any relationship to the mechanism(s) of liver metastasis remains to be elucidated.

Correlation between the sialylation of cell surface Thomsen-Friedenreich antigen and the metastatic potential of colon carcinoma cells in a mouse model.

The cell surface glycosylation profiles of a liver metastatic colon carcinoma variant cell line, SL4 cells previously selected from colon 38 cells in vivo for liver colonization were investigated. Flowcytometric analysis was performed with 7 plant lectins and 10 carbohydrate specific monoclonal antibodies. The results showed that peanut agglutinin (PNA), Sambucus nigra agglutinin, Ulex europeus agglutinin-I, anti-LeX, anti-LeY, and anti-Le(b) antibodies bound to the parental colon 38 cells but not to SL4 cells. Another variant cell line was selected in vitro for the paucity of cell surface PNA-binding sites using a magnetic cell sorter and was designated as 38-N4 cells. The binding profiles of plant lectins and carbohydrate-specific antibodies to 38-N4 cells were very similar to those of SL4 cells. After intrasplenic injections, metastatic ability of 38-N4 cells was higher than that of colon 38 cells. PNA binding to SL4 cells and 38-N4 cells was detected after sialidase treatment of these cells, indicating increased sialylation of Thomsen-Friedenreich antigen in these cells. The mRNA levels of sialyltransferases, ST3Gal I, ST3Gal II, ST6GalNAc I, and ST6GalNAc II, were compared. The level of ST3Gal II mRNA was elevated in both SL4 cells and 38-N4 cells, whereas the level of ST6GalNAc II mRNA was elevated in 38-N4 cells compared with colon 38 cells. According to the expression array analysis, there are other glycosyltransferase genes differentially expressed between SL4 and colon 38 cells, yet their involvement in the altered glycosylation in these cells is unclear.

Intercept-PCR, an improvement for elevating performance to find a new member of a certain gene family.

We have established a method by which the performance of reverse transcriptase coupled polymerase chain reaction (RT-PCR) for seeking a new gene is improved. The actual procedure is quite easy: it is only to add several specific oligonucleotides into the reaction mixture of the usual RT-PCR. To verify the effectiveness of this method is also easy: it is only to detect the PCR products in the preliminary experiment. The finding in the present study provides valuable information for gene cloning tactics.

Involvement of cytokines in the skin-to-lymph node trafficking of cells of the monocyte-macrophage lineage expressing a C-type lectin.

The mechanism by which dermal cells expressing a macrophage calcium-type lectin (MGL) trafficked to regional lymph nodes was investigated. Conditioned medium prepared from organ cultures of mouse skin sensitized with a mixture of acetone and dibutylphthalate was shown to decrease the number of MGL(+) cells in the dermis in ex vivo organ culture assays. In in vitro culture of sensitized skin, the loss of MGL(+) cells was abrogated by the addition to the culture medium of mAb against IL-1ss, while addition of recombinant IL-1ss to the medium in which untreated skin was cultured induced loss of MGL(+) cells. Intradermal injection of recombinant IL-1ss also resulted in a transient increase of MGL(+) cells in the T cell area of draining lymph nodes in vivo, indicating that IL-1ss is central in the entire process of MGL(+) cell trafficking to the lymph nodes. Supporting this is that cells producing IL-1ss were detected in the epidermis of cultured skin even early after sensitization. The possibility that IL-1ss simply down-regulates MGL expression was eliminated by Western blotting experiments with isolated MGL(+) cells treated with or without IL-1ss. IL-1alpha and tumor necrosis factor (TNF)-alpha were also able to induce migration of MGL(+) cells in the ex vivo assay in a manner akin to IL-1ss, and antibodies against them abrogated this. Isolated MGL(+) cells from skin cultured in type I collagen matrix in vitro displayed morphological changes upon exposure to IL-1ss, IL-1alpha or TNF-alpha, indicating that these cytokines exert a direct effect on these cells. Thus, pro-inflammatory cytokines, particularly IL-1ss, are produced at the site of skin sensitization and are involved in at least initiating the trafficking of cells expressing MGL to the lymph nodes.

Vital immunostaining of human gastric and colorectal cancers grafted into nude mice: a preclinical assessment of a potential adjunct to videoendoscopy.

Videoendoscopy has not significantly advanced diagnostic accuracy beyond that attainable by conventional fiberscopy, with respect to microcarcinomas of the digestive tract. We suspected that after the labeling of these lesions with an agent detectable by videoendoscope, digital processing of the images could facilitate endoscopic diagnosis of microcarcinomas. We have developed a novel antibody labeled with an indocyanine green (ICG) derivative that is evident by videoendoscope. However, the binding of such an exogenous antibody in vivo to tumor surfaces has not been described. In this preliminary study, after transplanting human gastric cancer or colorectal cancer into nude mice, we successfully bound the tumors in vivo with an anti-MUC1 mucin antibody, as subsequently confirmed by the performing of immunohistochemistry with a secondary antibody. The antibody labeled with an ICG derivative may therefore be clinically useful in detecting gastrointestinal microcarcinoma by videoendoscopy.

Migration of dermal cells expressing a macrophage C-type lectin during the sensitization phase of delayed-type hypersensitivity.

Dermal cells expressing a macrophage C-type lectin (mMGL) were previously suggested to migrate to regional lymph nodes during the sensitization phase of delayed-type hypersensitivity (DTH). The migration seemed to be induced by the solvent used to dissolve the antigen, and the DTH response was significantly enhanced by the migration. In this study, immunohistochemical analysis of skin after epicutaneous application of one of such solvents, a mixture of acetone and dibutylphthalate (AD), revealed a transient decrease in the number of mMGL-positive cells in the dermis. A similar decrease in this cell population was also observed in an ex vivo assay with skin explants excised from AD-treated sites. Conditioned medium from organ culture of AD-treated skin induced a similar decrease of mMGL-positive cells in untreated dermis, indicating the involvement of soluble factors. mMGL-positive cells seemed to represent a unique subpopulation of F4/80-positive dermal cells.

Involvement of cell surface glycans in adhesion of human colon carcinoma cells to liver tissue in a frozen section assay: role of endo-beta-galactosidase-sensitive structures.

Adhesion of human colon carcinoma variant cell lines expressing different levels of the cell surface sialyl Lewis X (sLeX) antigen to frozen sections of mouse liver was examined. KM12-HX cells that bound the monoclonal antibody (mAb) FH6 (anti-sLeX) and thus expressed a high level of sLeX demonstrated a greater degree of adhesion to liver sections than their low-binding counterparts, KM12-LX cells. The adhesion of KM12-HX cells to liver sections was partially blocked by mAb FH6, but not by another anti-sLeX mAb, KM93. The adhesion was Ca2+ dependent but was not inhibited by anti-E-selectin. Endo-beta-galactosidase treatment significantly reduced adhesion and resulted in the loss of cell surface binding sites for mAb FH6. O-linked oligosaccharides from KM12-HX cells incubated in the presence of p-nitrophenyl-N-acetylgalactosaminide were fractionated by a combination of gel filtration, anion exchange chromatography, and normal phase high-performance liquid chromatography. The structure of a mAb FH6-reactive and endo-beta-galactosidase-sensitive glycan was estimated by matrix-assisted laser desorption ionization time-of-flight mass spectrometry in a post source decay mode and by glycosidase digestions to be NeuAc alpha2-3Gal beta1-4GlcNAc beta1-3Gal beta1-4Glc-NAc beta1-3Gal beta1-4(+/-Fuc alpha1-3)GlcNAc beta1-6(NeuAc alpha2-3Gal beta1-3)GalNAc-pNP. Mild detergent lysates of mouse liver surface-labeled with sulfo-NHS biotin were incubated with glutaraldehyde-fixed monolayers of KM12-HX cells, and bound components were isolated after EDTA treatment. A Mr 49,000 component that bound only to KM12-HX cells and not to KM12-LX cells was identified.