Comorbidities and prediction of length of hospital stay

Columnar epithelium-lined esophagus (Barrett's esophagus) is an acquired disorder associated with a high incidence of adenocarcinoma of the lower esophagus. Columnar epithelium resembling intestinal metaplasia (IM) is especially important, since it is considered to be a premalignant condition. The aim of this study was to define the sulfated carbohydrate chain of mucin and its core peptide profile in Barrett's esophagus (BE) and to compare it with the profile in Barrett's adenocarcinoma and lower esophageal adenocarcinoma. The sulfated carbohydrate chain was not expressed in 16 specimens of normal esophageal epithelium, but in BE, it was expressed in 50% (8/16) of the specimens. This chain was detected in 100% (7/7) of esophageal adenocarcinoma specimens, including four cases of Barrett's adenocarcinoma. These data suggest that the sulfated carbohydrate chain may be associated with malignant phenotype of the esophagus. MUC1 core peptide was positive in 87% (13/15) of BE specimens and in 29% (2/7) of the esophageal adenocarcinoma specimens. MUC2 core peptide was present in 57% (8/14) and 43% (3/7) of these specimens, respectively. These data suggest that Barrett's epithelium, which is similar to IM, but not normal esophageal epithelium, expresses the sulfated sugar chain which is known to be present in gastric IM and colonic mucosa. However, there was no significant correlation between the expression of the sulfated sugar chain and the expression of either mucin core peptide MUC1 or MUC2. Thus, this carbohydrate chain may be expressed on as yet unidentified core proteins, other than MUC1 or MUC2 core peptide, in BE and esophageal adenocarcinoma. Identification of these proteins may be very important in helping to detect premalignant status in BE.     

Glutamine extraction by the gut is reduced in depleted [corrected] patients with gastrointestinal cancer

In order to investigate the expression of MUC5AC mucin in normal gastric mucosa and gastric carcinomas, we produced 3 monoclonal antibodies (MAbs) using a MUC5AC synthetic peptide. The immunohistochemical study was performed using one of these MAbs (CLH2) which reacted with the different designs of peptides based on the MUC5AC tandem repeat and with native and deglycosylated mucin extracted from gastric tissues. CLH2 immunoreactivity was restricted to foveolar and mucopeptic neck cells in normal gastric mucosa. No reactivity was observed in type-I intestinal metaplasia. Out of 66 gastric carcinomas, 42 (63.6%) expressed MUC5AC. Most diffuse carcinomas were positive (83.3%), whereas only 59.3% of intestinal and 40.0% of atypical carcinomas expressed MUC5AC (p < 0.05). Gastric carcinomas with mixed pattern showed immunoreactivity in diffuse areas and decreased immunoreactivity in intestinal areas. Every early gastric carcinoma expressed MUC5AC, in contrast to 58.6% of advanced carcinomas (p < 0.05). A trend toward decreased immunoreactivity was observed in deep areas of advanced carcinomas in comparison with the respective superficial areas. Taking together the specific staining of foveolar and mucopeptic neck cells and the absence of immunoreactivity in intestinal metaplasia, we conclude that MUC5AC expression may be used as a marker of gastric differentiation. This assumption is further supported by the finding of MUC5AC immunoreactivity in most diffuse carcinomas, which usually display morphologic and histochemical signs of gastric differentiation. The expression of MUC5AC in early gastric carcinomas, regardless of their histologic type, suggests that all gastric carcinomas retain at least some cells with a gastric phenotype during the first steps of neoplastic development.     

Aberrant expression of MUC5AC and MUC6 gastric mucin genes in colorectal polyps

Altered mucin glycosylation and the de novo appearance of gastric mucin antigens have been described in colonic adenomas. The purpose of our study was to determine if expression of the gastric mucin genes MUC5AC and MUC6 occurs in colorectal adenomas and whether this correlates with histopathologic criteria of malignant potential. Immunohistochemical staining using antibodies against MUC5AC and MUC6 tandem repeat synthetic peptides was performed on specimens of normal colon mucosa (n = 26), hyperplastic polyps (n = 9) and adenomatous polyps (n = 111). Mucin mRNA levels were determined using RNase protection assays using riboprobes corresponding to unique non-repetitive sequences. MUC5AC and MUC6 staining were rarely detected and of low intensity in normal colon and hyperplastic polyps. The number of immunoreactive polyps and intensity of MUC5AC and MUC6 staining were greatest in larger adenomas of moderate villous histology and dysplasia. MUC5AC and MUC6 staining tended to decrease in highly villous polyps with severe dysplasia. Increased MUC5AC mRNA levels were found in 26/45 of adenomas tested compared with 0/9 normal colon specimens. MUC6 mRNA levels were found in 20/45 of adenomas compared with 1/9 normal colon specimens. MUC5AC and MUC6 mRNA were present more frequently and at higher levels in polyps with intermediate stages of size, villous histology and dysplasia. We conclude that aberrant expression of MUC5AC and MUC6 mucin genes is likely responsible for an expanded repertoire of mucin antigen expression in colorectal neoplasia.     

The anti-MUC1 monoclonal antibody BCP8 can be used to isolate and identify putative major histocompatibility complex class I associated amino acid sequences

MHC class I molecules were isolated from the MUC1-positive human breast adenocarcinoma cell line MCF-7 by immunoaffinity using the panreactive anti-class I monoclonal antibodies (MAb) W6/32. Acid-eluted peptides from the class I molecules were separated twice by high-performance liquid chromatography and tested for reactivity with the MAb BCP8, which reacts with the minimal MUC1 core peptide sequence PDTRPA. A peak with strong and specific BCP8 reactivity was found in fractions eluting at 16.5-17.5 min. The protocol used for the MUC1+ pancreatic adenocarcinoma cell line CAPAN-1 (HLA.A2) was to perform sequential affinity purifications of class I molecules using MAb W6/32, followed by affinity purification of HLA.A2 molecules by the HLA.A2.1-specific MAb, MA2.1, and high-performance liquid chromatography fractionation of the acid-eluted material. A single peak with MAb BCP8 reactivity was noted at 18-19 min. The protocol for the MUC1+ breast adenocarcinoma cell line SKBr-3 (HLA.A11,B40), which used A11- and B40-specific MAbs, also resulted in the detection of BCP8-specific peaks at approximately 18-19 min. A preliminary mass spectral analysis of BCP8 affinity-purified class I associated material surprisingly revealed the presence of two 3-mer MUC1 amino acid sequences and one 6-mer sequence. A synthetic 9-mer MUC1 peptide, TSAPDTRPA, containing the isolated fragments was found to cause strong class I up-regulation in T2 cells as well as to serve as an epitope for CTL generated in a primary in vitro immune response. These studies suggest that MUC1-derived peptides are processed and presented in the context of MHC class I molecules on the surface of tumor cells and support the use of MAb BCP8 to further define MHC class I associated MUC1 motifs.     

Characterization of a new breast cancer-associated antigen and its relationship to MUC1 and TAG-72 antigens

We have characterized a new tumor-associated antigen defined by monoclonal antibody (MAb) generated against HMA-1 breast cancer cell line. MAb AM-1 was selected based on its preferential reactivity to breast cancer cells versus to normal or benign epithelial cells by immunofluorescence and immunohistochemical assays of cultured, or fresh specimens. AM-1 demonstrated strong reactivity to breast cancer cell lines including HMA-1, YMB-1-E, YMB-1 and MDA-MB-231 in flow cytometry. In immunoprecipitation, AM-1 recognized high molecular weight components of 160-210 kDa and > 370 kDa. Reactivity with HMA-1 cells was diminished markedly when treated by heat, protease or periodate, suggesting that the antigenic epitope is composed with carbohydrates and peptides. Enzyme digestion of precipitated antigens demonstrated that the antigen contains O-linked and N-linked carbohydrates with neuraminic acid structures. Furthermore, binding inhibition and sandwich ELISA assays using MAbs reactive with known breast cancer-associated antigens and synthetic MUC1 core peptide (PDTRPAPGSTAPPAHGVTSAPDTR) demonstrated that the antigen is distinct from CEA, TAG-72 or MUC1, while the antigen conjoins with MUC1 and TAG-72 as a trimmer form in HMA-1 cells. These results suggest that AM-1 recognizes a novel glycoprotein which is abundant in breast cancer, and may be utilized in the management of breast cancer patients.     

Suppression of sigma spindle electroencephalographic activity as a measure of transient arousal after spontaneous and occlusion-evoked sighs and startles

Gastric M1 mucin and the MUC5AC gene show a similar oncofetal expression in the colon. Our aim was to determine whether M1 mucin is the product of the MUC5AC gene. A recombinant baculovirus encoding the C-terminal portion of the MUC5AC gene as a fusion protein was isolated and the immunoreactivity of the recombinant mucin (rM) toward M1 antibodies studied. Chicken antibodies also were raised against purified rM. Besides its reactivity with L56/C, a serum recognizing the bacterially expressed MUC5AC gene product, rM was endowed with M1 immunoreactivity: (i) rM-expressing cells were stained specifically with anti-M1 serum and with the monoclonal antibody (MAb) 21M1, defining the M1-f epitope; (ii) both L56/C and anti-M1 antibodies recognized the same bands in immunoblots of rM-containing cell extracts; (iii) the 21M1 antibody reacted with rM in an immunoradiometric assay. Among the 7 M1 epitopes, M1-f was the only one encoded by the 3' portion of the MUC5AC gene. It was the only epitope detected in a native mucin M1-derived 170 kDa bromelain proteolytic fragment. Furthermore, the staining patterns of human tissues obtained with either anti-rM chicken antibodies or anti-M1 antibodies were identical. We conclude that M1 immunoreactivity is encoded at least in part by the MUC5AC gene.     

Expression of MUC1 and MUC2 mucins in gastric carcinomas: its relationship with the prognosis of the patients

Our previous immunohistochemical studies for the expression of MUC1 mucin antigen (which was detected by monoclonal antibody DF3) and MUC2 mucin antigen (which was detected by polyclonal antibody anti-MRP) in pancreatic and intrahepatic bile duct tumors demonstrated that invasive carcinoma with poor outcome showed a pattern of MUC1+ and MUC2- expression, whereas many of the noninvasive tumors with favorable outcome showed a pattern of MUC1- and MUC2+ expression. To clarify the relationship between the expression of these mucin antigens and the biological properties of gastric cancers, the expression of MUC1 and MUC2 mucin antigens was examined immunohistochemically in 136 patients with gastric cancer invading the submucosa or the deeper layer, and the survival of the antigen-positive and antigen-negative patient groups was compared using the Kaplan-Meier method. For MUC1 mucin expression, different glycoforms of MUC1 were examined using four monoclonal antibodies (NCL-MUC-1-CORE, DF3, MY.1E12, and HMFG-1). The patients with MUC1+ mucin antigen staining in the carcinoma showed significantly worse survival than those with MUC1- mucin antigen staining. In contrast, the patients with MUC2+ mucin antigen staining in the carcinoma showed significantly better survival than those with MUC2- mucin antigen staining. In conclusion, MUC1 antigen expression was associated with a poor outcome in patients with gastric cancer, irrespective of its glycosylation status, and MUC1 is thus considered to be a useful prognostic factor for poor outcome in patients. In contrast, MUC2 antigen expression is a prognostic factor associated with a favorable outcome in patients. In addition, combined evaluation of the MUC1 and MUC2 mucin staining is clinically useful to predict outcome in patients with gastric cancer.     

Selection of tumor antigens as targets for immune attack using immunohistochemistry: protein antigens

The relative expression of mucin antigens MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC5B, and MUC7 and glycoprotein antigens KSA, carcinoembryonic antigen, prostate-specific membrane antigen (PSMA), HER-2/neu, and human chorionic gonadotropin-beta on different cancers and normal tissues is difficult to determine from available reports. We have compared the distribution of these antigens by immunohistology on a broad range of malignant and normal tissues. MUC1 expression was most intense in cancers of breast, lung, ovarian, and endometrial origin; MUC2 was most intense in cancers of colon and prostate origin; and MUC5AC was most intense in cancers of breast and gastric origin. MUC4 was intensely expressed in 50% of cancers of colon and pancreas origin, and MUC3, MUC5B, and MUC7 were expressed in a variety of epithelial cancers, but not so intensely. KSA was intensely and uniformly expressed on all epithelial cancers; carcinoembryonic antigen was expressed in most cancers of breast, lung, colon, pancreas, and gastric origin; and PSMA was expressed only in cancers of prostate origin. Human chorionic gonadotropin-beta was expressed on the majority of sarcomas and cancers of breast, lung, and pancreas origin, although intense staining was not seen. Staining on normal tissues was restricted to one or many normal epithelial tissues ranging from MUC3, MUC4, and PSMA, which were expressed only on epithelia of pancreas, stomach, and prostate origin, respectively, to MUC1 and KSA, which were expressed on most normal epithelia. Expression was restricted to the secretory borders of these epithelia while stroma and other normal tissues were completely negative. These results plus the results of the two previous papers (S. Zhang et al, Int. J. Cancer, 73: 42-49, 1997; S. Zhang et al., Int. J. Cancer, 73: 50-56, 1997) in this series provide the basis for selection of multiple cell surface antigens as targets for antibody-mediated attack against these cancers.     

Bioaccumulation of heavy metals by fimbrial designer adhesins

BACKGROUND & AIMS: Alterations in the production of epithelial mucins have been correlated with advanced tumor stage in the colon, but direct evidence for a role of specific mucin genes in liver metastasis is lacking. The current study was designed to establish more directly the role of MUC2 in colon cancer metastasis. METHODS: MUC2 levels were manipulated in highly metastatic human colon cancer cells using eukaryotic expression constructs designed to express a portion of MUC2 complementary DNA in antisense orientation. To assess the effect of MUC2 levels on metastatic potential, liver colonization was assessed in athymic mice after splenic-portal inoculation. RESULTS: Stable integration of the MUC2 antisense construct into metastatic colon cancer cells (LS LiM6) resulted in an 80% reduction in MUC2-specific messenger RNA and a concomitant decrease in MUC2 apomucin protein. This reduction was associated with a 50% reduction in synthesis of mature glucosamine-labeled mucin, almost complete inhibition of secretion of sialyl-LeX and sialyl-Tn antigens, and a 40% decrease in binding of colon cancer cells to E-selectin. Reduction in MUC2 levels was associated with a marked decrease in liver colonization. CONCLUSIONS: This study provides direct evidence that MUC2 plays an important role in colon cancer metastasis.     

Differential expression of human high-molecular-weight salivary mucin (MG1) and low-molecular-weight salivary mucin (MG2)

Two distinct mucin components of saliva, MG1 and MG2, have been identified based on chemical composition and molecular weights (high and low, respectively) in saliva. With the aim of characterizing the expression pattern of salivary mucins, we have prepared monoclonal antibodies (MAbs) directed against the peptide core of MG1 and against a synthetic peptide derived from the MG2 (MUC7) sequence. MAb PANH2 raised against partially deglycosylated MG1 stained a high-molecular-weight smear in Western blots of partially purified MG1. PANH2 binding was increased by deglycosylation with trifluoromethanesulfonic acid as well as with subsequent periodate treatment, and was eliminated by pronase treatment, strongly suggesting that MAb PANH2 was directed to a peptide epitope of MG1. MAb PANH3 raised against a synthetic peptide derived from the MG2 (MUC7) sequence reacted with the native molecule and stained a narrow smear of ca. 200,000 to 210,000 in Western blots of concentrated saliva and a lower-molecular-weight smear of trifluoromethanesulfonic-acid-treated MG2. Immunohistology on frozen sections of human salivary glands showed that MAb PANH2 selectively labeled mucous cells, whereas MAb PANH3 labeled subpopulations of serous cells. Double-direct immunofluorescence staining with PANH2 and PANH3 demonstrated that the staining patterns were non-overlapping. The development of these antibody probes will facilitate studies of mucin expression in diseases of salivary glands.     

Human single-chain Fv antibodies to MUC1 core peptide selected from phage display libraries recognize unique epitopes and predominantly bind adenocarcinoma

The tumor-associated antigen MUC1 is overexpressed and underglycosylated in human adenocarcinomas of diverse origins, such as breast, ovary, and colon. We isolated and describe five human single-chain (sc) Fv antibodies specific for the MUC1 variable number of tandem repeats region isolated by in vitro selection from a large naive phage antibody library containing over 6 x 10(9) different scFv antibodies. A synthetic biotinylated 100-mer peptide corresponding to five tandem repeats of the MUC1 peptide core was used for selection. Two of the antibodies were highly specific for MUC1 as judged by ELISA and flow cytometry. In immunohistochemistry, antibody clone 10A stained MUC1 in the cytoplasm and membrane of adenocarcinoma cells of breast and ovary, whereas in normal epithelium, only cytoplasmic or no staining was observed. With antibody clone 10B, staining was less pronounced and was not always membrane associated in adenocarcinoma. Determination of the fine specificity of 10A and 10B using a novel "indirect epitope fingerprinting" ELISA showed that both antibodies recognize unique epitopes that have not been described for hybridoma-derived anti-mucin antibodies of mouse origin. The selected human antibodies, like many of the murine MUC1 antibodies, recognize epitopes on the protein core of MUC1 that are abundantly present in the underglycosylated form of cell surface mucin on adenocarcinoma. The best human scFv, clone 10A, appears to distinguish normal cells from adenocarcinoma cells, which makes it an attractive candidate for use in antibody-based tumor targeting.     

Electrophysiologic effects of civamide (zucapsaicin) on canine cardiac tissue in vivo and in vitro

Mucins are highly expressed in many different human cancers and numerous murine monoclonal antibodies (MAbs) to human mucins, particularly Mucin 1 (MUC1), have been produced. However, no such antibodies to murine mucin 1 (muc1) have been described and we now describe 6 different antibodies produced to murine muc1 and to human MUC1 cytoplasmic tail, either by immunising rats, or muc1 o/o mice with synthetic peptides or a fusion protein composed of glutathione-s-transferase (GST) linked to the tandem repeat region of muc1. The antibodies to both the extracellular tandem repeat region and to the cytoplasmic tail were found to react with mucin-containing murine tissues such as breast, stomach, colon, ovary, kidney and pancreas, and the staining patterns were similar to those found in humans. The reagents reacted specifically with muc1 peptides and tissues; however, some cross reactivity with other mucin-derived peptides was noted, particularly those containing the amino acid sequence TSS. Three different epitopes (TSS, TAVLSGTS and LSGTSSP) of the M30, M70 and MFP25 MAbs were detected. Of interest was the finding that some of the antibodies reacted with murine lymphocytes; it was not clear whether these reactions were due to mucin 1 on mouse lymphocytes (MUC1 was considered to be absent from human lymphocyte), or due to cross reaction with a sialic adhesion molecule on lymphocytes. The antibodies should prove valuable reagents when studying differentiation and expression in murine glandular tissues and the ontogeny of mucin-secreting tumours.     

Structure, function and gene expression of epithelial mucins

In this review the main characteristics, i.e., structure, function and gene expression, of the different mucins are discussed. Mucin-type molecules consist of a core protein moiety (apomucin) where a number of carbohydrate chains are attached to serines and threonines by glycosidic bonds. O-linked carbohydrates form up to 80% of the molecule and the length of the glucidic side chains varies from one to more than 20 residues. At least eight mucin-like genes have been isolated so far, and the main characteristic is the presence of a central domain composed of a variable number of "tandem repeats". The sequence homology of the central domain among the different members of the mucin-type family is limited, indicating that this internal domain is unique for each mucin. Thanks to the integrated results of genetic, immunological and biochemical studies, it is now possible to identify eight apomucin genes, namely MUC1, MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC6 and MUC7. MUC1 is the best characterized mucin and it is expressed on the apical surface of most polarized epithelial cells. The MUC1 gene has been cloned and sequenced. The MUC2 gene encodes a typical secretory gel-forming mucin which represents the predominant form in human intestinal and colon tissues. Another intestinal mucin is MUC3. The MUC4, MUC5AC and MUC5B genes have been isolated from a bronchial tissue cDNA library. The MUC4 and MUC5AC genes are mainly expressed in the respiratory tract, in gastric and reproductive mucosa, while MUC5B is highly detectable only in the bronchial glands. The MUC6 gene is expressed by gastric tissue and, recently, MUC7 has been cloned and sequenced using a salivary cDNA library.     

MUC1 is a novel marker for the type II pneumocyte lineage during lung carcinogenesis

Abnormalities in mucin-type glycoprotein expression have been documented in a variety of cancers, identifying these molecules as targets for immunologically based therapies and prognostic/diagnostic assays. We examined the expression of the membrane-bound MUC1 mucin in normal, histologically atypical, and neoplastic lung to determine its potential contribution to lung carcinogenesis. In vivo, intense MUC1 immunoreactivity was present in normal type II pneumocytes as well as in a range of atypical lesions derived from type II cells and >60% of primary and metastatic non-small cell lung cancers. Expression was not associated with altered survival, although it was highly correlated with the adenocarcinoma histology. A carcinogenesis model using 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone-exposed hamsters revealed that MUC1 mRNA increased prior to the histological appearance of tumors. In vitro studies using MUC1 expressing non-small cell lung cancer cell lines revealed that differentiation away from a type II cell lineage was associated with dramatic down-regulation of MUC1. We propose that MUC1 is a powerful new marker for the type II pneumocyte cell lineage that allows us to follow the type II pneumocyte lineage during the process of lung carcinogenesis.     

Mucin antigen expression in gastric carcinomas of young and old adults

The striking differences in the histological features of gastric cancers in young and old adults have been thought to be related to differences in the biological behavior of these cancers. Recently, a new grading system (Goseki's classification) showed that the prognosis of the patient is particularly related to the mucin content of the carcinoma. In this study, we examined differences in mucin antigen expression in cancers from young and old adults and whether antigen expression is related to the clinical outcome. The expression of two mucin core proteins (DF3 antigen [MUC1 gene product] and MRP antigen [MUC2 gene product] and a mucin-type carbohydrate antigen [sialosyl-Tn; STn]) was examined immunohistochemically in gastric cancers from 69 young adults (30 to 39 years of age) and 110 old adults (80 to 89 years of age). The incidence rates of the three histological types (tubular adenocarcinoma, poorly differentiated adenocarcinoma, and signet-ring cell carcinoma) were different in the young and old adults. However, among the mucin antigens examined, only DF3 showed significantly higher frequency of expression in the old adults, and the difference was seen only in tubular adenocarcinomas (young, 43%; old, 68%) and poorly differentiated adenocarcinomas (young, 19%; old, 49%). In these two histological types, there was no difference in the frequency of MRP or STn expression between the young and old adults, although the old adults showed a high incidence of intestinal metaplasia that was positive for both antigens. Signet-ring cell carcinomas showed no significant difference in expression rates of the three antigens in young and old adults, but there were significantly higher expression rates in young patients for both MRP (young, 67%; old, 65%) and STn (young, 71%; old, 57%) and a lower rate of DF3 expression (young, 0%; old, 14%). In both young and old adults, the patients with DF3-positive carcinomas showed significantly poorer survival than those without DF3 expression, whereas there was not significant difference in the survival of the patient groups with positive and negative MRP or STn reactivity. In conclusion, the expression of DF3 was influenced by the age of patients and was related to the outcome. In contrast, MRP and STn expression was related more to the histological pattern of the tumor than to the age of the patient and did not correlate with the outcome.     

Synergistic neutralization of a chimeric SIV/HIV type 1 virus with combinations of human anti-HIV type 1 envelope monoclonal antibodies or hyperimmune globulins

A panel of 14 human IgG monoclonal antibodies (MAbs) specific for envelope antigens of the human immunodeficiency virus type 1 (HIV-1), 2 high-titer human anti-HIV-1 immunoglobulin (HIVIG) preparations, and 15 combinations of MAbs or MAb/HIVIG were tested for their ability to neutralize infection of cultured human T cells (MT-2) with a chimeric simian immunodeficiency virus (SHIV-vpu+), which expressed HIV-1 IIIB envelope antigens. Eleven MAbs and both HIVIGs were neutralizing. When used alone, the anti-CD4-binding site MAb b12, the anti-gp41 MAb 2F5, and the anti-gp120 MAb 2G12 were the most potent. When combination regimens involving two MAbs targeting different epitopes were tested, synergy was seen in all paired MAbs, except for one combination that revealed additive effects. The lowest effective antibody concentration for 50% viral neutralization (EC50) and EC90 were achieved with combinations of MAbs b12, 2F5, 2G12, and the anti-V3 MAb 694/98D. Depending on the combination regimen, the concentration of MAbs required to reach 90% virus neutralization was reduced approximately 2- to 25-fold as compared to the dose requirement of individual MAbs to produce the same effect. Synergy of the combination regimens implies that combinations of antibodies may have a role in passive immunoprophylaxis against HIV-1. The ability of SHIV to replicate in rhesus macaques will allow us to test such approaches in vivo.     

Expression cloning of gastric mucin complementary DNA and localization of mucin gene expression

BACKGROUND & AIMS: Secretory mucins play an important role in gastric cytoprotection and are derived from a heterogeneous family of genes. The aim of this study was to determine the specific type and location of mucin gene expression in the human stomach. METHODS: Expression cloning was performed by screening a human gastric complementary DNA expression library with antisera against deglycosylated gastric mucin. RNA analysis and immunohistochemistry were used to quantify and localize mucin gene expression. RESULTS: Sequencing of positive clones revealed two clones containing tandem repeats. The first contained a 169-amino acid repeat and was named MUC6 (as previously described). The second contained the same 8-amino acid repeat consensus sequence (APTTSTTS) as complementary DNAs previously isolated from a tracheobronchial complementary DNA library and was labeled MUC5 (or MUC5AC). RNA analysis indicated that the gastric epithelium contains high levels of MUC5 and MUC6 messenger RNA with little or no MUC2, MUC3, and MUC4 messenger RNA. Immunohistochemical analysis showed that surface mucous cells of the cardia, fundus, and antrum expressed MUC5 peptide. In contrast, MUC6 peptide expression was limited to mucous neck cells of the fundus, antral-type glands of the antrum and cardia, and Brunner's glands of the duodenum. CONCLUSIONS: MUC5 and MUC6 represent major secretory mucins in the stomach and are localized to distinct cell types.     

Novel monoclonal antibodies to putative selectin carbohydrate ligands that inhibit selectin binding to myeloid cells

Four newly developed monoclonal antibodies (MAbs) are characterized using flowcytometry, enzyme-linked immunoadsorbent assay (ELISA), immunoprecipitation and Western blots, carbohydrate epitope mapping, glycosidase cleavage, and competition binding assays. Their effects on selectin binding to myeloid cells was tested. These MAbs react only with myeloid cells. MAbs CI-1, BU60, and HIM95 recognize epitopes expressed by CD11/CD18 (beta2) integrins, while HI247 and CSLEX1 do not. The epitopes require Lewis x [Galbeta1-4 (Fucalpha1-3)GlcNAc] based on reactivity with oligosaccharide-polyacrylamide-biotin or oligosaccharide-BSA conjugates. MAb HI247 recognizes a related structure, sialyl-Lewis x, NeuAcalpha2-3GaLbeta1-4(Fucalpha1-3)GlcNAc. The three MAbs against Lewis x show some minor differences in their reactivity such as recognizing their antigens on CD11/CD18 integrins after endo-beta-galactosidase treatment and recognizing free Lewis x. The hydroxyl group on C-3 of the terminal galactose is important for recognition by MAb CI-1, BU60, and HIM95 as its substitution with sulfo group of sialic acid abolishes the binding of these MAbs. The C-3 sialic acid is crucial for the binding of MAb HI247. Its replacement by sulphate or its cleavage by sialidase eliminates recognition by this MAb. MAbs HI247 and CSLEX-1 did not react in ELISA with immobilized CD11/CD18, suggesting that the majority of sialyl Lewis x on CD11/CD18 molecules may have sialic acid 6-linked rather than 3-linked to galactose. Unexpectedly, MAb BU60 inhibited binding of P-selectin mu chain chimera to HL-60 or U937 cells, while CI-1, HIM95 and three other defined anti-Lewis x MAbs (6C7, M6-1 and LeuM1) did not. MAb HI247 inhibited binding of both E- and P-selectin chimeras to these cell lines more effectively than several characterized MAbs (CSLEX-1, FH6, HECA-452) to sialyl Lewis x and related oligosaccharides. Certain combinations of these anticarbohydrate MAbs had additive inhibitory effects on selectin binding, suggesting a potential application of these new MAbs in cell adhesion/migration and tumor metastasis studies.     

Ulcerative colitis and coexisting colorectal cancer: recurrence rate after restorative proctocolectomy

BACKGROUND: The association between mucosal ulcerative colitis (MUC) and adenocarcinoma is well established. METHODS: Records of patients who had undergone restorative proctocolectomy with ileal pouch-anal anastomosis (IPAA) from 1983 through 1992 were examined. Of these, 604 had MUC and 27 (4.3%) had MUC with coexisting cancer. Patients were surveyed annually for recurrent disease. Pouch function and quality of life were evaluated with a questionnaire and physical examination. RESULTS: The duration of disease was longer (p = 0.001) in patients with cancer (16.1 +/- 8.0 years) than in those without cancer (9.1 +/- 7.1 years), although the mean age at diagnosis of MUC was the same. Of the 27 patients, 20 had colon cancer and seven had rectal cancer. Multicentricity was found in seven (25.9%) patients. Using the TNM staging classification, 14 patients (51.8%) had stage 1 cancer, eight (29.6%) had stage 2, four (14.8%) had stage 3, and one (3.8%) had stage 4. The patient with stage 4 cancer died 5 months after surgery and was excluded from the follow-up analysis. During a mean follow-up time of 4.3 +/- 2.6 years, cancer recurred in two of the remaining 26 patients (7.7%). In one patient, a local recurrence was found 8 months after surgery, and distant metastases were found in the other patient 35 months after surgery. Both recurrences were in patients with colon cancer. Two of the 26 patients died; one death was related to cancer recurrence (3.8%). Pouch function is good to excellent in all surviving patients. CONCLUSIONS: Restorative proctocolectomy for patients with MUC and coexisting colorectal cancer can be performed with a favorable prognosis and function. It is appropriate for curative intent, given that an adequate margin without tumor is obtained.