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OBJECTIVE: Asthma is the most common chronic airway inflammatory disease. Sirtuin 1 (SIRT1) exerts a crucial effect on regulating chronic inflammatory responses. Therefore, this study aims to explore the effect of SIRT1 on the pathogenesis of asthma. MATERIALS AND METHODS: Serum level of SIRT1 in asthma patients and healthy controls was detected by Western blot. Correlation between SIRT1 level and pulmonary function in asthma patients was analyzed. Subsequently, asthma model in mouse was established. Primary airway epithelial cells were extracted from asthma mice and control mice to detect SIRT1 level. Furthermore, relative levels of Akt and interleukin 6 (IL-6) were detected in 16HBE cells. Regulatory effects of Akt on SIRT1 in 16HBE cells were determined as well. RESULTS: SIRT1 was highly expressed in serum of asthma patients, which was negatively correlated with FEV1/FVC (r=-0.27, **p<0.01). Both mRNA and protein levels of SIRT1 were downregulated in primary airway epithelial cells extracted from asthma mice compared with those from controls. SIRT1 knockdown in 16HBE cells upregulated IL-6 expression, which was reversed by Akt inhibitors. CONCLUSIONS: SIRT1 regulates IL-6 level via Akt pathway, thereafter affecting pulmonary function in asthma patients. [ABSTRACT FROM AUTHOR]

OBJECTIVE: Cytokine-induced killer cells (CIK) is a type of immune cell with antitumor activity induced by a variety of cytokines. Regulatory T cells (Treg) is a T cell subgroup featured as immunosuppressive function. Existing CIK cultivation system may inevitably induce Treg. Forkhead box protein 3 (Foxp3) is an essential transcription factor for Treg function. This study aimed to investigate the effects of CIK on the leukemia cell HL-60. MATERIALS AND METHODS: This work silenced Foxp3 expression on the basis of CIK induction, aiming to investigate its killing effect on HL-60 cells. Peripheral blood mononuclear cells were separated and differentiated to CIK in vitro. CD3+CD56+ and CD4+CD25+Foxp3+ Treg cells were detected by flow cytometry. CIK cells were co-cultured with HL-60 cells under the effectortarget ratio at 20:1, 10:1, and 5:1, respectively. The killing activity of CIK on HL-60 cells was determined by CCK-8 assay. RESULTS: The ratio of CD3+, CD3+CD8+, and CD3+CD56+ cells gradually increased during CIK induction. Foxp3 interference significantly reduced Treg cell ratio on the 7th day (p < 0.05). Treg cell ratio was significantly lower in Foxp3 interference group at 1.62% ± 0.07% compared with control (p < 0.05). The killing activity of CIK on HL-60 cells enhanced following the increase of effectortarget ratio. Interference of Foxp3 significantly elevated the killing activity of CIK on HL-60 cells with effector-target ratio dependence (p < 0.05). CIK can effectively suppress HL-60 cell growth. Treg significantly inhibited the anti-tumor effect of CIK. CONCLUSIONS: Interference of Foxp3 expression significantly declined Treg level and attenuated its suppression impact on CIK, thus enhancing the killing effect of CIK on HL-60 cells.. [ABSTRACT FROM AUTHOR]

Doxorubicin (DOX) selection of CCRF-CEM leukaemia cell line resulted in multidrug resistance (MDR) CEM/A7R cell line, which overexpresses MDR, 1 coded P-glycoprotein (Pgp). Here, we report for the first time that oncoprotein Cripto, a founding member of epidermal growth factor-Cripto-FRL, 1-Criptic family is overexpressed in the CEM/A7R cells, and anti-Cripto monoclonal antibodies (Mab) inhibited CEM/A7R cell growth both in vitro and in an established xenograft tumour in severe combined immunodeficiency mice. Cripto Mab synergistically enhanced sensitivity of the MDR cells to Pgp substrates epirubicin (EPI), daunorubicin (DAU) and non-Pgp substrates nucleoside analogue cytosine arabinoside (AraC). In particular, the combination of anti-Cripto Mab at less than 50% of inhibition concentrations with noncytotoxic concentrations of EPI or DAU inhibited more than 90% of CEM/A7R cell growth. Cripto Mab slightly inhibited Pgp expression, and had little effect on Pgp function, indicating that a mechanism independent of Pgp was involved in overcoming MDR. We demonstrated that anti-Cripto Mab-induced CEM/A7R cell apoptosis, which was associated with an enhanced activity of the c-Jun N-terminal kinase/stress-activated protein kinase and inhibition of Akt phosphorylation, resulting in an activation of mitochondrial apoptosis pathway as evidenced by dephosphorylation of Bad at Ser136, Bcl-2 at Ser70 and a cleaved caspase-9.British Journal of Cancer (2007) 96, 918–927. doi:10.1038/sj.bjc.6603641 www.bjcancer.com Published online 6 March 2007 [ABSTRACT FROM AUTHOR]

CD46 (membrane cofactor protein; MCP) is ubiquitously expressed on nucleated human cells; it has a protective function, binding C3b and C4b, which are then cleaved by serum factor I. CD46 molecules (55 000-65 000 MW) have four short consensus repeats (SCR): the function of SCR-1 and -2 is unknown; SCR-3 and 4 bind C3b and C4b. These are succeeded by the STP region, which can contain three separate regions (STP-A, -B, -C) rich in serine, threonine and proline and which are heavily glycosylated, succeeded by transmembrane and cytoplasmic tail regions (of which there are several). Multiple isoforms exist due to the different splicing of exons: STP-A and -B can thus be present or absent. So far these products can only be detected separately by polymerase chain reaction (PCR) and RNA studies; we now describe their detection by anti-peptide antibodies. Peptides whose sequences corresponded with those of STP-A and STP-B were synthesized and used for the immunization of mice; although they differ in only seven of 21 amino acids, monocional antibodies (mAb) that reacted specifically with STP-A but not with STP-B, and mAb that reacted specifically with STP-B but not with STP-A, were produced; these reacted specifically with native CD46 on human tissues and cell lines. STP-A mAb reacted with tissues in which STP-A RNA had been found, some leukaemias and cell lines; in normal tissue expression was mainly found in the intestine (large and small) and salivary gland. Anti-STP-B reacted with most tissues and cell lines. The antibodies should be of use in defining the expression and function of CD46 in different tissues. [ABSTRACT FROM AUTHOR]

Monoclonal anti-mucine antibodies BC1, BC2 and BC3 produced using human milk fat globule membrane react with a synthetic peptide p1-24 (PDTRPAPGSTAPPAHGVTSAPDTR) representing the repeating amino acid sequence of the mucin core protein. The minimum epitope recognized by these three monoclonal antibodies (mAb) in p1-24 was contained in the five amino acids APDTR. To analyse the variation of position of the epitope, various modifications of the APDTR sequence were made by synthesizing peptides and testing by direct binding and inhibition enzyme-linked immunosorbent assays. Firstly, peptides p13-32 and C-p13-32, in which the epitope APDTR was placed in the middle instead of the C-terminal as in p1-24, were examined. These peptides had a greater reaction with mAb BC1, BC2 and BC3 compared with the reaction with p1-24. Secondly, A-p1-24 and TSA-p1-24 were made wherein two APDTR epitopes were present—these peptides were shown to bind two IgG antibody molecules. Finally, the contribution of each amino acid in the APDTR epitope was studied using the pepscan polyethylene rods, making all 20 of the amino acid substitutions in each position for SAPDTR (the minimum epitope APDTR with an adjacent amino acid S). In the 120 peptides examined there were some 'permissible' substitutions in A, D and T but not in P or R for BCi and BC2; there were more 'permissible' substitutions for BC3; different substitution patterns were found with each antibody and some substitutions gave an increased reaction compared with the native peptide SAPDTR. The studies are of value in analysing the reaction of antibodies with epitopes expressed in breast cancer and in determining the antigenicity of synthetic peptides. [ABSTRACT FROM AUTHOR]

This study reports the characterization of a breast cancer-associated antigen identified by murine monoclonal antibody (MoAb) RCC-I (formerly called 24-1 71). Immunoperoxidase staining indicated that RCC-1 recognized an antigen highly expressed in malignant tumours of breast origin, and no reactivity was noted with connective tissue, muscle or lymph nodes, which is an important consideration in its successful use in immunolymphoscintigraphy. The RCC-1 was shown to consist of 94 000 dalton disulfide-bonded dimers which were shown to be different from the transferrin receptor. In addition, the antibody RCC-1 did not react with components of human milk or with an antigenic peptide derived from the core protein of a mammary mucin. Chemical treatment and enzymatic digestion suggested that the epitope recognized by antibody RCC-1 was protein as it was resistant to neuraminidase and periodate treatment but was sensitive to trypsin. The RCC-1-defined antigen detects a novel breast cancer associated antigen. [ABSTRACT FROM AUTHOR]

Three murine monoclonal antibodies (BC1, BC2 and BC3) were developed against human milk fat globule membrane (HMFGM). By immunoperoxidase staining, it was found that the antigenic determinants had a predominant distribution in breast cancer tissue. In addition, the antibodies reacted preferentially with mucin derived from human milk rather than that derived from the breast cancer cell line ZR75; they also recognized polymorphic high molecular weight components (MW≥230 000) in serum and in human milk fat globule membrane. Thus the antibodies appear to react with a component of the family of mucins found in breast cancer and human milk and it appears likely that at least part of each epitope is protein in nature. Antibodies BCI, BC2 and BC3 recognized related but not identical epitopes, and they appear to be co-expressed on the same molecules as 3E1-2-defined antigen (mammary serum antigen, MSA) which is also a member of the family of breast cancer-rotated mucin. However, the 3E1-2 epitope is distinct and non-cross-reactive with those described for BCI, BC2 and BC3. The BC2 and BC3 defined epitopes were examined for their value in serum assays. Immunoassay was developed with a combination of two antibodies, using antibody BC3 for antigen capture and antibody BC2 or 3E1-2 for antigen detection and gave reasonable sensitivity (∼85%) and specificity (∼95%) in such serum tests for breast cancer. In a limited study, these tests appeared to complement the MSA test in the detection of breast cancer. [ABSTRACT FROM AUTHOR]

Multiple genes coding for human mucins have been identified (MUC 1-5) and here monoclonal antibodies (MoAb) to a gastrointestinal mucin-MUC2 are examined. The antibodies were made to a synthetic peptide representing a single repeat in the core protein of the variable number of tandem repeat region. Using the six-mer overlapping peptides synthesized on polyethylene pins, different binding sites were detected by five anti-MUC 2 MoAbs. These contained amino acids: STTT, PTT, GTQTP, TPTP and PTTT (one antibody), and TPTPT. The repeat region of M UC2 essentially is hydrophobic, but contain useful immunogenic sites. This information will be useful for studying the structure and function of MUC2. [ABSTRACT FROM AUTHOR]

Purpose To determine the prognostic significance and long term survival of breast cancer patients with overexpression of the epidermal growth factor Cripto. Methodology 120 formalin fixed paraffin embedded breast cancer specimens were constructed on a tissue microarray and detection of Cripto carried out by immunohistochemical staining. Patients were treated between 1989 and 1995 and the median follow up was 125 months. We examined the association of Cripto positivity with age, menopausal status, grade and size of tumour, lymph node status, tumour type, ER/PR/HER2 status, Ki67 and Nottingham Prognostic Index (NPI). Results 48% of patients were Cripto positive. We demonstrated a significant association between overexpression of Cripto and NPI (p < 0.01), grade of tumour (p < 0.01), progesterone receptor (p = 0.02), Ki 67 (p = 0.02), tumour type (p = 0.04) and most importantly overall survival (p = 0.0003). Cox regression analysis revealed Cripto to be an independent prognostic variable for survival – HR 2.79 (95% CI 1.20–6.50). Conclusion Overexpression of Cripto is associated with high grade poor prognostic breast cancer and a significantly decreased patient survival. Future research is required to confirm these findings and to develop an anti-Cripto humanised antibody for clinical use. [ABSTRACT FROM AUTHOR]

Legionella is a common intracellular parasitic bacterium that infects humans via the respiratory tract, causing Legionnaires' disease, with fever and pneumonia as the main symptoms. The emergence of highly virulent and azithromycin-resistant Legionella pneumophila is a major challenge in clinical anti-infective therapy. The CRISPR-Cas acquired immune system provides immune defense against foreign nucleic acids and regulates strain biological functions. However, the distribution of the CRISPR-Cas system in Legionella and how it regulates gene expression in L. pneumophila remain unclear. Herein, we assessed 915 Legionella whole-genome sequences to determine the distribution characteristics of the CRISPR-Cas system and constructed gene deletion mutants to explore the regulation of the system based on growth ability in vitro , antibiotic sensitivity, and intracellular proliferation of L. pneumophila . The CRISPR-Cas system in Legionella was predominantly Type II-B and was mainly concentrated in the genome of L. pneumophila ST1 strains. The Type II-B CRISPR-Cas system showed no effect on the strain's growth ability in vitro but significantly reduced resistance to azithromycin and decreased proliferation ability due to regulation of the lpeAB efflux pump and the Dot/Icm type IV secretion system. Thus, the Type II-B CRISPR-Cas system plays a crucial role in regulating the virulence of L. pneumophila . This expands our understanding of drug resistance and pathogenicity in Legionella , provides a scientific basis for the prevention of Legionnaires' disease outbreaks and the rational use of clinical drugs, and facilitates effective treatment of Legionnaires' disease., Competing Interests: The authors declare no conflict of interest.

MUC1 is a potential target in breast cancer immunotherapy as MUC1 is overexpressed in breast cancer, and is absent or expressed in low level in normal mammary gland. In addition, MUC1 is mostly aberrantly underglycosylated in cancer and the antigens on the cancer surface are different from normal cell. Therefore targeting MUC1 for cancer immunotherapy can exploit the difference between cancer and normal cells, and eliminating the cancerous cells while leaving the normal mammary cells unharmed. This review will focus on the recent advance of MUC1 breast cancer immunotherapy currently being investigated.

Introduction: Cripto is a founding member of the EGF-CFC family, and plays an important role in tumourigenesis, tumour cell proliferation and migration. We aimed to determine the significance of Cripto expression on the survival of patients with breast cancer., Methods: Immunohistochemical detection of Cripto was performed by using mAb C13 on 120 formalin-fixed paraffin-embedded breast tumour specimens in tissue microarrays. This cohort comprises a series of 120 patients with primary operable breast cancer diagnosed between 1989 and 1995, retrieved from the Concord Repatriation General Hospital breast carcinoma database., Results: Using a cutoff value of 80%, Cripto overexpressed in 57 of the 120 (47.5%) patients. We found significant associations between overexpression of Cripto and the Nottingham Prognostic Index (NPI, p<0.01), histological grade (p<0.01), pathological tumour type (p=0.04), PR (p=0.02) as well as Ki-67 (p=0.02). Univariate analysis reveals that there is a significant correlation between overexpression of Cripto and survival (p=0.0003). Cox regression analysis indicates that the overexpression of Cripto is an independent prognostic factor in breast cancer (HR 2.79, 95%CI 1.20-6.50)., Conclusion: The unique epitope recognized by mAb C13 is overexpressed on breast tumour tissues. In this series of invasive breast cancers, overexpression of Cripto was more often found in high grade and poor prognosis tumours compared to low grade and good prognosis breast cancers. Moreover, overexpression of Cripto was significantly associated with decreased patient survival.

The MUC1 mucin (CD227) is a cell surface mucin originally thought to be restricted to epithelial tissues. We report that CD227 is expressed on human blood dendritic cells (DC) and monocyte-derived DC following in vitro activation. Freshly isolated murine splenic DC had very low levels of CD227; however, all DC expressed CD227 following in vitro culture. In the mouse spleen, CD227 was seen on clusters within the red pulp and surrounding the marginal zone in the white pulp. Additionally, we confirm CD227 expression by activated human T cells and show for the first time that the CD227 cytoplasmic domain is tyrosine-phosphorylated in activated T cells and DC and is associated with other phosphoproteins, indicating a role in signaling. The function of CD227 on DC and T cells requires further elucidation.

Mucins are of major interest in cell biology, not only are they highly over-expressed in many adenocarcinomas (up to 40-fold increase), but also have important physiological function, and probably more to be determined (1-3). There is much information available on mucins - doubtless because of their unusual structure being heavily glycosylated, but also containing a repeat region rich in the amino acids serine, threonine and proline. This repeat region confers high immunogenicity of the mucins, and as a result, many antibodies (Abs) have been made to mucins of different species (4). Furthermore, the production of Abs led to the cloning of the cDNAs and armed with these reagents (antibodies, cDNA and genomic structures), advances in the knowledge of the structure and function of mucins has been rapid, together with the development of transgenic and gene knockout animals for biological studies (1-9). Here we describe monoclonal antibodies (Mabs) made to the different mucins, including Mucins 1-4, concentrating on human Mucin 1 (MUC1), to variants of MUC1, to regions outside the VNTR of MUC1, mouse Mucin1 (muc1), unusual features and cross reactions of anti-MUC1 Mabs and Abs made by patients in clinical trials. We will especially describe the Mabs produced in our laboratory.

Background: Natural antibodies that react with galactose-alpha(1,3)galactose [galalpha(1,3)gal] carbohydrate epitopes exist in humans and Old World primates because of the inactivation of the alpha1,3-galactosyltransferase (alpha1,3GT) gene in these species and the subsequent production of antibodies to environmental microbes that express the galalpha(1,3)gal antigen. The Gal knockout (Gal o/o) mouse, produced by homologous disruption of the alpha1,3GT gene, spontaneously makes anti-galalpha(1,3)gal antibodies and can be used to study the genetic control of humoral immune responses to this carbohydrate epitope., Methods: Six hybridomas that produce monoclonal antibodies (mAbs) to galalpha(1,3)gal were generated in Gal o/o mice. The mAbs were tested to characterize the binding activity with flow cytometry using pig aortic endothelial cells and ELISA with galalpha(1,3)gal carbohydrates. The VH and VK genes of these hybridomas were cloned, sequenced, and analyzed., Results: The mAbs showed distinct patterns of antibody binding to galalpha(1,3)gal antigens. The VH genes that encode the mAb binding activity were restricted to a small number of genes expressed in their germline configuration. Four of six clones used closely related progeny of the same VH germline gene (VH441). Comparison of the mouse gene VH441 to the human gene IGHV3-11, a gene that encodes antibody activity to galalpha(1,3)gal in humans, demonstrates that these two genes share a nonrandom distribution of amino acids used at canonical binding sites within the variable regions (complimentary determining regions 1 and 2) of their immunoglobulin VH genes., Conclusions: These results demonstrate the similarity of the Gal o/o mice and humans in their immune response to galalpha(1,3)gal epitopes. Gal o/o mouse can serve as a useful model for examining the genetic control of antibody/antigen interactions associated with the humoral response to pig xenografts in humans.

Cystic fibrosis knockout mice (cftr(-/-)) die prematurely of obstruction of the intestine which may result from accumulation of dehydrated glycoconjugate-containing mucus. We noted an increase in the specific activity of [(14)C]glucosamine-labeled high-molecular weight glycoconjugates, probably mucin, in the lumen of the intestine of cftr(-/-) (homozygous) mice compared to cftr(+/+) (wild-type) and cftr(+/-) (heterozygous) mice and a decrease in the turnover of glycoconjugates of several organs of the cftr(-/-) mice. No difference in the anionic composition of secreted intestinal glycoconjugates was detected and no difference in the amount of mucin 1 (Muc1) was found in the small intestine, colon, pancreas, and lungs of the different genotypes. In addition, the spleen of the cftr(-/-) mice was significantly smaller than that of control mice and the small intestine and colon were, respectively, longer and shorter compared to control mice. These results indicate modified glycoconjugate metabolism in cystic fibrosis knockout mice and morphologic changes to the spleen and intestine where the latter modifications are possibly related to the intestinal malabsorption associated with cystic fibrosis., (Copyright 2001 Academic Press.)

Monoclonal antibodies (mAbs) were produced to human haptoglobin by immunising with fecal extracts from patients with colon cancer. An enzyme-linked immunosorbent assay was developed with one of the mAbs (FE14.1), and its ability to diagnose colorectal carcinoma evaluated. Patients with colorectal cancer were positive (43/46 = 93.5%) compared to normal individuals (4/211 = 1.9%). The assay has a specificity 93.5% and sensitivity 98.1% and has several advantages over current fecal occult blood tests. The test is potentially useful for bowel cancer diagnosis and to quantitate the level of haptoglobin in other body fluids such as urine and in effusions.

Mucin-1 (MUC-1), which is overexpressed in more than 90% of human breast cancers, is a potential target for immunotherapy. To develop a mouse model appropriate for the immunotherapy of human cancer, mouse mucin-1 (muc-1) fusion protein, containing ten tandem repeats, was made and used to immunize C3H/HeOuj mice, which supposedly have a high incidence of breast cancer. C3H/HeOuj mice were injected eight times with 5 microg oxidized mannan muc-1-glutathione-S-transferase (MMFP) with or without cyclophosphamide, which is used to increase cellular immunity. At 80 age weeks, only 12.1% (4 of 33) mice of the untreated C3H/HeOuj mice had mammary tumors. The reason for the low incidence of breast cancer in these mice is not known, but all the mammary tumors were MUC-1+ breast adenocarcinomas and were transplantable to C3H/HeOuj mice. The incidence was 11.4% (4 of 35) in mice injected with MMFP: 38.2% (13 of 34) in mice given cyclophosphamide; and 14.3% (2 of 14) in mice treated with glutathione-S-transferase. That is, cyclophosphamide increased the incidence of mammary tumors, and metastases were found in only these mice. Fewer tumors (6 of 34 or 17.6% compared with 13 of 34 or 38.2% with cyclophosphamide only) occurred in the group immunized with MMFP and cyclophosphamide. Mice immunized with MMFP had high levels of muc-1 antibodies and cellular immune responses (the frequency of the precursor of the cytotoxic Tlymphocyte cell was 1 of 40,000 to 1 of 100,000), which were not found in control groups. The occurrence of muc-1 immunity, particularly the presence of large amounts of anti-mucin-1 antibodies, had no effect on tumor incidence. Thus, the immunization with murine muc-1 reduced the tumor incidence in only cyclophosphamide-treated mice and led to strong muc-1 antibody production and to cellular responses. These findings have implications for human tumor immunotherapy in which strong antibody and weak cellular responses are to be expected and, indeed, have been found.

MUC1 mucin is a transmembrane glycoprotein that is highly expressed in various cancer cell lines and is also present in most of the glandular epithelial cells including the airway. Although the presence of numerous phosphorylation sites in its cytoplasmic domain suggests its potential role as a receptor, the unavailability of a ligand for MUC1 mucin has limited our understanding of its function. In this paper, we tried to circumvent this problem by constructing a chimeric receptor containing the cytoplasmic domain of MUC1 mucin, which can be phosphorylated on activation. To this end, we constructed a chimeric plasmid vector (pCD8/MUC1) by replacing the extracellular and transmembrane domains of human MUC1 mucin with those of human CD8. Transient transfection of the vector into COS-7 cells resulted in expression of the chimeric receptor on the surface of the COS-7 cells as judged by immunologic assays with various antibodies as well as by fluorescence-activated cell-sorting analysis. Treatment of the transfected COS-7 cells with an anti-CD8 antibody resulted in a significant increase in phosphorylation of tyrosine moieties of the chimeric receptor. This chimeric receptor will serve as a powerful tool in elucidating the signaling mechanism as well as the functional role of MUC1 mucin in the airway.

Analyses of MUC1-specific cytotoxic T cell precursor (CTLp) frequencies were performed in mice immunized with three different MUC1 vaccine immunotherapeutic agents. Mice were immunized with either a fusion protein comprising MUC1 and glutathione S-transferase (MUC1-GST), MUC1-GST fusion protein coupled to mannan (MFP) or with a recombinant vaccinia virus expressing both MUC1 and interleukin-2. Mouse strain variations in immune responsiveness have been observed with these vaccines. We have constructed mice transgenic for the human MUC1 gene to study MUC1-specific immune responses and the risk of auto-immunity following MUC1 immunization. Transgenic mice immunized with MUC1 were observed to be partially tolerant in that the MUC1-specific antibody response is lower than that observed in syngeneic but non-transgenic mice. However, a significant MUC1-specific CTLp response to all three vaccines was observed, indicating the ability to overcome T cell, but to a lesser extent B cell, tolerance to MUC1 in these mice. Histological analysis indicates no evidence of auto-immunity to the cells expressing the human MUC1 molecule. These results suggest that it is possible to generate an immune response to a cancer-related antigen without damage to normal tissues expressing the antigen.

Using 2 anti-mucin 1 (MUC1) monoclonal antibodies (MAbs), DF3 and BCP8, we examined MUC1 expression immunohistochemically in 192 esophageal squamous-cell carcinomas (SCCs). In normal squamous epithelium of the esophagus, DF3 was not expressed, but BCP8 was expressed on the cell membrane, mainly in the surface layer. In esophageal SCCs, DF3 and BCP8 were expressed mainly on the cell membrane of SCC cells, but also in the cytoplasm in several cases. To analyze the correlation of MUC1 expression and the prognosis of the patients, the 192 cases were divided into 2 groups: high-expression group (HEG, > 50% of the neoplastic cells stained) and low-expression group (LEG, < 50% of neoplastic cells stained). DF3-HEG (24 patients) showed a significantly poorer survival rate than DF3-LEG (168 patients), whereas there was no significant difference in survival between BCP8-HEG (43 patients) and BCP8-LEG (149 patients). Also, in the analysis of 162 patients with advanced stage (submucosal or deeper invasion) to exclude the influence of low expression of DF3 and BCP8 in 30 patients with early stage (up to the level of muscularis mucosae), DF3-HEG (24 patients) showed significantly poorer survival than DF3-LEG (138 patients), whereas there was no significant difference in survival between BCP8-HEG (42 patients) and BCP8-LEG (120 patients). The results of our study on esophageal SCC suggest that the expression of sialyl oligosaccharides detected by DF3 is related to poor prognosis.

Peptides bound in the groove of MHC class I molecules and detected by CTLs are not normally accessible to Ab. We now report that MUC1 peptides that are bound within the groove of MHC class I molecules (H2 and HLA) and that can be detected by CTLs can also be detected by anti-MUC1 Abs. mAbs to the middle and C-terminal regions of the class I-associated peptides but not to the N terminus were able to react with MUC1 peptides bound to H2Kb and HLA-A*0201, and only to the mid-region for H2Db, by flow cytometry and also to block CTL activity. Molecular modeling showed that the N terminus is buried (and not accessible), whereas the midpeptide residues form a loop and the C terminus is free, making these two regions accessible to Ab. The findings demonstrate for the first time that peptides associated with class I molecules can be detected by anti-peptide Abs.

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.

It is desirable to be able to produce either T1 or T2 responses and we have found that, in mice, mannose--coupled antigens stimulated T2 type responses antibodies and CTLs, whereas if oxidized, mannose--coupled antigens stimulated T1 responses little antibody and a potent CTL response. In addition, the cytokine profiles support the T1rT2 differentiation with these immunizations, in that oxidized mannan antigen gives IFNg, IL-2 and IL-12 production, whereas in the absence of oxidization, IL-4 and not the other cytokines is produced. A number of antigens have been examined--particularly Mucin 1 and the delivery method using mannose may be applicable to the other antigens.

Mucin 1 (MUC1) is a large complex glycoprotein that is highly expressed in breast cancer, and as such could be a target for immunotherapy. In mice, human MUC1 is highly immunogenic, particularly when conjugated to mannan, where a high frequency of CD8(+) MHC-restricted cytotoxic T lymphocytes is induced, accompanied by tumor protection. On this basis, a clinical trial was performed in which 25 patients with advanced metastatic carcinoma of breast, colon, stomach, or rectum received mannan-MUC1 in increasing doses. After 4 to 8 injections, large amounts of IgG1 anti-MUC1 antibodies were produced in 13 out of 25 patients (with antibody titers by ELISA of 1/320-1/20,480). Most of the antibodies reacted to the epitopes STAPPAHG and PAPGSTAP. In addition, T cell proliferation was found in 4 out of 15 patients, and CTL responses were seen in 2 out of 10 patients. Mannan-MUC1 can immunize patients, particularly for antibody formation, and to a lesser extent, cellular responses. It remains to be seen whether such responses have antitumor activity.

In a comprehensive study, we examined the expression of the membrane and secretory mucins MUC1 and MUC3, respectively, in normal and neoplastic gastrointestinal and breast epithelia before and after specific alterations of their glycan structures by neuraminidase, alpha-fucosidase, or carbohydrate-specific periodate oxidation. MUC1 mRNA was also identified in normal colorectal tissues by in situ hybridization. The data revealed that normal colorectal epithelia express both MUC1 mRNA and protein, which were detectable after periodate oxidation with all tested MUC1-specific antibodies. During tumorigenesis in the colon, MUC1 became recognizable without periodate treatment concomitantly with highly dysplastic lesions and the malignant state. In the breast, in which MUC1 is detectable with most antibodies in normal epithelium as well as in carcinomas, staining could be enhanced by pretreatment with periodate and casually by enzyme treatments. MUC3 was detectable in normal and neoplastic colorectal tissues and was more intensely stained after periodate oxidation. It was absent in normal breast even after pretreatment but was expressed in seven of 20 breast carcinomas. Therefore, incomplete glycosylation, abnormal distribution, and ectopic expression of mucins are characteristics of malignancy. Periodate oxidation may be widely applicable to immunohistochemistry for examining changes in glycosylation and for detecting antigens masked by glycans.