Immunoreactivity of Lewis blood group and mucin peptide core antigens: correlations with grade of dysplasia and malignant transformation in the colorectal adenoma-carcinoma sequence

Previous studies on the immunoreactivity of various mucin peptide and carbohydrate antigens in neoplastic colorectal tissues led to at least in part contradictory results. Therefore, we investigated a series of 42 adenomas and 44 carcinomas applying monoclonal antibodies (mabs) directed against Lewis blood group antigens (sialyl-Le(a), Le(x), sialyl-Le(x), Le(y)) as well as mucin peptide cores (MUC1, MUC2 and MUC5AC) by immunohistochemistry. A statistically significant positive correlation between the development of high-grade dysplasia in colorectal adenomas and the immunoreactivity of Le(y) and MUC1 epitopes was observed, whereas MUC2 exhibited a significant negative correlation. The reactivity of the other epitopes did not show an association with the progression of malignant transformation. Colorectal carcinomas were subdivided according to their histopathological subtype. The immunohistochemical staining resulted in a significantly stronger MUC2 reactivity of mucinous vs. tubular adenocarcinomas. Immunoreactivity of the MUC1-specific mab, which does not react with the fully glycosylated peptide core, showed a statistically non-significant inverse tendency, whereas all carbohydrate antigens displayed a strong expression in both tumor subtypes. Furthermore, correlations between mucin peptide and carbohydrate epitope labelling were evaluated. Progression of the adenoma-carcinoma sequence was accompanied by an increase of Le(y) as well as MUC1 antigen and an increase of all Lewis antigens compared to MUC2 immunoreactivity. On the other hand, mucinous carcinomas exhibited an inverse pattern. In conclusion, these results demonstrate that Le(y) and MUC1 immunoreactivity correlate with malignant transformation in the colorectum, whereas MUC2 represents a marker for low-grade dysplasia and the subtype of mucinous carcinomas.     

Mucin 21 in esophageal squamous epithelia and carcinomas: analysis with glycoform-specific monoclonal antibodies

Monoclonal antibodies (mAbs) against mucin 21 (MUC21), a human counterpart of mouse epiglycanin/Muc21, were prepared using human embryonic kidney 293 cells transfected with MUC21 as the immunogen. The specificity of these mAbs was examined by flow cytometry, immunoprecipitation and western blotting focusing on the differential glycosylation of MUC21 expressed in variant Chinese hamster ovary (CHO) cells (ldlD cells and Lec2 cells) and CHO-K1 cells. One of these mAbs, heM21D, bound to both the unmodified core polypeptide of MUC21 and MUC21 attached with N-acetylgalactosamine (Tn-MUC21). Six antibodies, including mAb heM21C, bound to MUC21 with Tn, T or sialyl-T epitopes but not the unmodified core polypeptide of MUC21. Esophageal squamous carcinomas and adjacent squamous epithelia were immunohistochemically examined for the binding of these mAbs. MUC21 was expressed in esophageal squamous epithelial cells, and its O-glycan extended forms were observed in the luminal portions of squamous epithelia. As revealed by the binding of mAb heM21D and the absence of reactivity with mAb heM21C, esophageal squamous carcinoma cells produce MUC21 without the attachment of O-glycans. This is the first report to show that there is a change in the glycoform of MUC21 that can be used to differentiate between squamous epithelia and squamous carcinoma of the esophagus. Thus, these antibodies represent a useful tool to characterize squamous epithelial differentiation and carcinogenesis.     

A straightforward protocol for the preparation of high performance microarray displaying synthetic MUC1 glycopeptides

Background

Human serum MUC1 peptide fragments bearing aberrant O-glycans are secreted from columnar epithelial cell surfaces and known as clinically important serum biomarkers for the epithelial carcinoma when a specific monoclonal antibody can probe disease-relevant epitopes. Despite the growing importance of MUC1 glycopeptides as biomarkers, the precise epitopes of most anti-MUC1 monoclonal antibodies remains unclear.

Methods

A novel protocol for the fabrication of versatile microarray displaying peptide/glycopeptide library was investigated for the construction of highly sensitive and accurate epitope mapping assay of various anti-MUC1 antibodies.

Results

Selective imine-coupling between aminooxy-functionalized methacrylic copolymer with phosphorylcholine unit and synthetic MUC1 glycopeptides-capped by a ketone linker at N-terminus provided a facile and seamless protocol for the preparation of glycopeptides microarray platform. It was demonstrated that anti-KL-6 monoclonal antibody shows an extremely specific and strong binding affinity toward MUC1 fragments carrying sialyl T antigen (Neu5Acα2,3Galβ1,3GalNAcα1→) at Pro-Asp-Thr-Arg motif when compared with other seven anti-MUC1 monoclonal antibodies such as VU-3D1, VU-12E1, VU-11E2, Ma552, VU-3C6, SM3, and DF3. The present microarray also uncovered the occurrence of IgG autoantibodies in healthy human sera that bind specifically with sialyl T antigen attached at five potential O-glycosylation sites of MUC1 tandem repeats.

Conclusion

We established a straightforward strategy toward the standardized microarray platform allowing highly sensitive and accurate epitope mapping analysis by reducing the background noise due to nonspecific protein adsorption.

General significance

The present approach would greatly accelerate the discovery research of new class autoantibodies as well as the development of therapeutic mAbs reacting specifically with disease-relevant epitopes.     

Outer membrane protein III of Neisseria gonorrhoeae: variations in biological properties of antibodies directed against different epitopes

Monoclonal antibodies (mAbs) have been raised to gonococcal outer membranes. A panel of six mAbs was identified by several criteria as reacting with outer membrane protein III (P.III). Competitive radioimmunoassays showed that the mAbs could be grouped into three pairs recognizing different epitopes on P.III. These epitopes are equally present on all pathogenic Neisseria. The mAbs demonstrated differing protective effects in model systems. Those directed against one epitope were particularly effective in protecting Chang conjunctiva epithelial cells against gonococcal challenge. mAbs against this epitope and another promoted complement-mediated bactericidal activity, while those directed against the third epitope were ineffective. Thus the biological effects of mAbs directed against P.III vary according to the epitope recognized.     

Binding patterns of DTR-specific antibodies reveal a glycosylation-conditioned tumor-specific epitope of the epithelial mucin (MUC1)

Glycosylation determines essential biological functions of epithelial mucins in health and disease. We report on the influence of glycosylation of the immunodominant DTR motif of MUC1 on its antigenicity. Sets of novel glycopeptides were synthesized that enabled us to examine sole and combined effects of peptide length (number of repeats) and O-glycosylation with GalNAc at the DTR motif on the binding patterns of 22 monoclonal antibodies recognizing this motif. In case of unglycosylated peptides almost all antibodies bound better to multiple MUC1 tandem repeats. Glycosylation at the DTR led to enhanced binding in 11 cases, whereas 10 antibodies were not influenced in binding, and one was inhibited. In nine of the former cases both length and DTR glycosylation were additive in their influence on antibody binding, suggesting that both effects are different. Improved binding to the glycosylated DTR motif was exclusively found with antibodies generated against tumor-derived MUC1. Based on these data a tumor-specific MUC1 epitope is defined comprising the ...PDTRP... sequence in a particular conformation essentially determined by O-glycosylation at its threonine with either GalNAcalpha1 or a related short glycan. The results can find application in the field of MUC1-based immunotherapy.     

Expression of core 2 beta-1,6-N-acetylglucosaminyltransferase in a human pancreatic cancer cell line results in altered expression of MUC1 tumor-associated epitopes.

Many tumor-associated epitopes possess carbohydrate as a key component, and thus changes in the activity of glycosyltransferases could play a role in generating these epitopes. In this report we describe the stable transfection of a human pancreatic adenocarcinoma cell line, Panc1-MUC1, with the cDNA for mucin core 2 GlcNAc-transferase (C2GnT), which creates the core 2 beta-1,6 branch in mucin-type glycans. These cells lack endogenous C2GnT activity but express a recombinant human MUC1 cDNA. C2GnT-transfected clones expressing different levels of C2GnT were characterized using monoclonal antibodies CC49, CSLEX-1, and SM-3, which recognize tumor-associated epitopes. Increased C2GnT expression led to greatly diminished expression of the CC49 epitope, which we identified as NeuAcalpha2,6(Galbeta1,3)GalNAcalpha-Ser/Thr in the Panc1-MUC1 cells. This was accompanied by the emergence of the CSLEX-1 epitope, sialyl Lewis x (NeuAcalpha2,3Galbeta1,4(Fucalpha1,3)GlcNAc-R), an important selectin ligand. Despite this, however, the C2GnT transfectants could not bind to selectins. Increased C2GnT expression also led to masking of the SM-3 peptide epitope, which persisted after the removal of sialic acid, further suggesting greater complexity of the core 2-associated O-glycans on MUC1. The results of this study suggest that C2GnT could play a regulatory role in the expression of certain tumor-associated epitopes.     

Mapping of the 45M1 epitope to the C-terminal cysteine-rich part of the human MUC5AC mucin

Mucins are large glycoproteins protecting mucosal surfaces throughout the body. Their expressions are tissue-specific, but in disease states such as cystic fibrosis, inflammation and cancer, this specificity can be disturbed. MUC5AC is normally expressed in the mucous cells of the epithelia lining the stomach and the trachea, where it constitutes a major component of the gastric and respiratory mucus. A number of mAbs have been raised against the gastric M1 antigen, an early marker for colonic carcinogenesis. Several of these mAbs recognize epitopes present on MUC5AC, suggesting that MUC5AC is the antigen. However, some of the mAbs raised against the gastric M1 antigen are widely used as antibodies against MUC5AC, despite the fact that their specificity for MUC5AC has not been clearly shown. In this study, we have tested the reactivity of the latter antibodies against a recombinantly expressed C-terminal cysteine-rich part of human MUC5AC. We demonstrate for the first time that the widely used mAb 45M1, as well as 2-12M1 and 166M1, are true antibodies against MUC5AC, with epitopes located in the C-terminal cysteine-rich part of the mucin.     

Significance of Monoclonal Antibodies against the Conserved Epitopes within Non-Structural Protein 3 Helicase of Hepatitis C Virus

Nonstructural protein 3 (NS3) of hepatitis C virus (HCV), codes for protease and helicase carrying NTPase enzymatic activities, plays a crucial role in viral replication and an ideal target for diagnosis, antiviral therapy and vaccine development. In this study, monoclonal antibodies (mAbs) to NS3 helicase were characterized by epitope mapping and biological function test. A total of 29 monoclonal antibodies were produced to the truncated NS3 helicase of HCV-1b (T1b-rNS3, aa1192–1459). Six mAbs recognized 8/29 16mer peptides, which contributed to identify 5 linear and 1 discontinuous putative epitope sequences. Seven mAbs reacted with HCV-2a JFH-1 infected Huh-7.5.1 cells by immunofluorescent staining, of which 2E12 and 3E5 strongly bound to the exposed linear epitope ¹²³¹PTGSGKSTK¹²³⁹ (EP05) or core motif ¹³⁷³IPFYGKAI¹³⁸⁰ (EP21), respectively. Five other mAbs recognized semi-conformational or conformational epitopes of HCV helicase. MAb 2E12 binds to epitope EP05 at the ATP binding site of motif I in domain 1, while mAb 3E5 reacts with epitope EP21 close to helicase nucleotide binding region of domain 2. Epitope EP05 is totally conserved and EP21 highly conserved across HCV genotypes. These two epitope peptides reacted strongly with 59–79% chronic and weakly with 30–58% resolved HCV infected blood donors, suggesting that these epitopes were dominant in HCV infection. MAb 2E12 inhibited 50% of unwinding activity of NS3 helicase in vitro. Novel monoclonal antibodies recognize highly conserved epitopes at crucial functional sites within NS3 helicase, which may become important antibodies for diagnosis and antiviral therapy in chronic HCV infection.     

Dissection of the poly(Glu60 Ala30 Tyr10) (GAT)-specific T-cell repertoire in H-2I k mice

Twenty-five allospecific monoclonal antibodies (mAb), produced in the A. TH. A.BY, or B10.S (7R) anti-A.TL combinations, were shown to recognize determinants organized in four spatially distinct polymorphic regions on the same I-Ak-encoded molecule(s). These reagents were used to assess the recognition of the class II major histocompatibility complex (MHC) determinants in a series of GAT-reactive A.TL T-cell clones exhibiting various restriction specificity or alloreactivity patterns. Of the proliferative responses of 13 cloned T cells, 12 responses were found to be inhibited similarly by the same set of mAbs.A hierarchy in the blocking effects of these reagents that could be correlated with the spatial organization of their determinants was observed. (i) All the mAbs defining the epitope region I (i.e., recognizing public Ia.1- or Ia.17-like determinants, presumably expressed on the A beta subunit) and some of those identifying new public determinants in the epitope region II profoundly inhibited these T-cell responses. (ii) Intermediate blocking was observed when mAbs recognizing public determinants in the epitope region III were used. (iii) Finally, among the mAbs that identified the epitope group IV, the Ia.19-specific mAb 39.J was inhibitory, whereas mAbs directed against private Ia.2-like determinants were not. By contrast, the GAT-specific proliferative response of the T-cell clone AT-20.1, which recognized its nominal antigen in an extensively cross-reactive MHC-restricted fashion, could only be inhibited by a subset of the mAbs recognizing epitopes in groups I and II, but not by those recognizing epitopes in groups III and IV. It was also shown that the same subset of I-Ak-and I-Au-reactive mAbs displayed similar blocking effects on the proliferation of two T-cell clones exhibiting dual specificity for I-Ak- and I-Au-restricting and/or I-Ak- and I-Au-alloactivating determinants. Finally, all the cloned T-cell responses examined were found to be inhibited by rat mAbs against the LFA.1 molecule or the murine equivalent of the human OKT4 differentiation antigen. These studies suggest that class II specific mAbs can impair proliferation of cloned T-cells by a mechanism(s) other than the masking of the T-cells' restriction determinants per se.     

Properties of anti-gp41 core structure antibodies, which compete with sera of HIV-1-infected patients

To determine the correlation between the immunoreaction against the core structure of human immunodeficiency virus type (HIV-1) transmembrane protein gp41 epitopes and the disease progression, it is essential to evaluate the anti-core structure antibody epitopes and the humoral immunity against the epitopes. For this purpose we evaluated monoclonal antibodies (mAbs) against the gp41 core structure such as mAbs 50.69, 98.6 and T26, by Western blotting (WB) and flow cytometry. WB showed mAbs 50.69 and 98.6 bound to both monomeric and oligomeric gp41, and mAb T26 exclusively bound to oligomeric gp41. We evaluated the sera from Pneumocystis pneumonia patients (PCP; n=7) and long-term survivors (LTS; n=7). Competition assay with sera and mAbs for binding to H9 cells infected with HIV-1 IIIB virus was done using flow cytometry. The results revealed that PCP sera as well as LTS sera inhibited the binding of all the three mAbs, and the PCP sera inhibited mAb T26 binding more efficiently than LTS. Therefore, PCP patients retain competing immunity to antibodies against not only the shared epitopes of the core structure (binding sites of mAbs 50.69 and 98.6) but also against oligomeric gp41 specific epitope (binding site of mAb T26).     

Various stages of schistosoma express Lewis(x), LacdiNAc, GalNAcbeta1-4 (Fucalpha1-3)GlcNAc and GalNAcbeta1-4(Fucalpha1-2Fucalpha1-3)GlcNAc carbohydrate epitopes: detection with monoclonal antibodies that are characterized by enzymatically synthesized neoglycoproteins

We report here that fucosylated epitopes such as Lewis(x), LacdiNAc, fucosylated LacdiNAc (LDN-F) and GalNAcbeta1-4(Fucalpha1-2Fucalpha1-3)GlcNAc (LDN-DF) are expressed by schistosomes throughout their life cycle. These four epitopes were enzymatically synthesized and coupled to bovine serum albumin to yield neoglycoproteins. Subsequently these neoglycoproteins were used to probe a panel of 188 monoclonal antibodies obtained from infected or immunized mice, in ELISA and surface plasmon resonance analysis. Of these antibodies, 25 recognized one of the fucosylated structures synthesized, indicating that these structures are immunogenic during infection. The MAbs identified could be subdivided in four different groups based on the recognition of either the Lewis(x)-, the LacdiNAc-, the LDN-DF-, or both the LDN-F- and LDN-DF epitope. These monoclonal antibodies were then used to investigate the localization of the fucosylated epitopes in various stages of Schistosoma mansoni using indirect immunofluorescence. Lewis(x)epitopes were mainly found in the gut and on the tegument of adult worms, on egg shells, and on the oral sucker of cercariae. The LacdiNAc epitope was expressed on the tegument of adult worms, on miracidia, and on the oral sucker of cercariae. In contrast, LDN-DF epitopes were mainly present in the excretory system of adult worms, on miracidia and on whole cercariae. These also stained positive with the LDN-F/LDN-DF epitope antibodies, while whole parenchyma reacted characteristically only with the latter antibodies. The identification of different carbohydrate structures in various stages of schistosomes may lead to a better understanding of the function of glycans in the immune response during infection.     

Aberrantly glycosylated MUC1 is expressed on the surface of breast cancer cells and a target for antibody-dependent cell-mediated cytotoxicity

Protein glycosylation often changes during cancer development, resulting in the expression of cancer-associated carbohydrate antigens. In particular mucins such as MUC1 are subject to these changes. We previously identified an immunodominant Tn-MUC1 (GalNAc-α-MUC1) cancer-specific epitope not covered by immunological tolerance in MUC1 humanized mice and man. The objective of this study was to determine if mouse antibodies to this Tn-MUC1 epitope induce antibody-dependent cellular cytotoxicity (ADCC) pivotal for their potential use in cancer immunotherapy. Binding affinity of mAb 5E5 directed to Tn-MUC1 was investigated using BiaCore. The availability of Tn-MUC1 on the surface of breast cancer cells was evaluated by immunohistochemistry, confocal microscopy, and flow cytometry, followed by in vitro assessment of antibody-dependent cellular cytotoxicity by mAb 5E5. Biacore analysis demonstrated high affinity binding (K_D?=?1.7 nM) of mAb 5E5 to its target, Tn-MUC1. Immunolabelling with mAb 5E5 revealed surface expression of the Tn-MUC1 epitope in breast cancer tissue and cell lines, and mAb 5E5 induced ADCC in two human breast cancer cell lines, MCF7 and T47D. Aberrantly glycosylated MUC1 is expressed on the surface of breast cancer cells and a target for antibody-dependent cell-mediated cytotoxicity suggesting that antibodies targeting glycopeptide epitopes on mucins are strong candidates for cancer-specific immunotherapies.     

Immunodetection and localization of protein(s) related to retinal S-antigen (arrestin) in kidney.

S-antigen (arrestin) is a cytosolic protein which regulates phototransduction in retinal rods. A protein immunologically related to S-antigen was identified in fractions from soluble extract of bovine kidney enriched by gel filtration or by immunoaffinity chromatography using a polyclonal antibody to retinal S-antigen. On immunoblots, this protein was recognized by a panel of monoclonal antibodies (mAbs S2D2, S1A3 and S9E2) directed against different S-antigen epitopes and displayed the same apparent molecular mass (48 kDa) as retinal S-antigen. All three mAbs revealed a specific immunoreactivity by indirect immunocytochemical technique on rat kidney sections. The three mAbs recognized some but not all glomerular cells, identified as epithelial cells by immunoelectron microscopy using the mAb S9E2. Both mAbs S2D2 and S1A3 gave a diffuse cytoplasmic staining in all tubule cells. Proximal tubule cells exhibited a weak immunoreactivity, whereas distal and collecting tubule cells were strongly labeled. In contrast, the mAb S9E2 immunoreaction was restricted to a cell subpopulation from distal and collecting tubules corresponding to intercalated cells identified by immunoelectron microscopy. With the mAb S9E2, the labeling of proximal tubule cells was localized in the apical region of the cytoplasm. These results suggest that two or more 48-kDa proteins immunologically cross-reactive with retinal S-antigen are present in kidney. The observed pattern of distribution is in keeping with the hypothesis that such proteins could play a role in the regulation of G-protein-related receptors present in renal glomerulus and tubule epithelial cells.     

The Human Endometrium Expresses the Glycoprotein Mucin-1 and Shows Positive Correlation for Thomsen-Friedenreich Epitope Expression and Galectin-1 Binding

Mucin 1 (MUC1) is a glycoprotein in human endometrium and is abundant at the luminal epithelial surface in the receptive phase. It has a highly glycosylated ecto-domain that contains keratan sulfate chains, that disappears at the time of implantation. In addition, the glycoforms on MUC1 differ in fertile and infertile women. Therefore the aims of this study were investigations on glycosylation of MUC1 with the Thomsen-Friedenreich (TF) epitope on normal human endometrium throughout the menstrual cycle and binding of galectin-1 on the TF epitope in the endometrium and the expression of galectin-1 on the human oocyte. Human endometrial tissue was obtained from 54 premenopausal patients and was immunohistochemically analyzed with monoclonal antibodies against MUC1, TF epitope, galectin-1, and biotinylated galectin-1. In addition, human oocytes were analyzed for TF, galectin-1 expression, and galectin-1 binding. We identified a significant upregulation of MUC1 and TF epitope and, in addition, galectin-1 binding in glandular epithelium and epithelial apical surface tissue from proliferative to secretory phase. With double staining experiments, we identified a coexpression of TF and MUC1 in the early secretory phase and galectin-1 binding to TF during the same period of time. In addition we identified TF epitope and galectin-1 expression plus binding on the human oocyte and irregularly fertilized oocytes. Upregulation of TF epitope on the glandular epithelium and epithelial apical surface tissue in the secretory phase and binding of galectin-1 at the same time show the possibility of galectin-1–mediated trophectoderm binding to the endometrium within the window of implantation. (J Histochem Cytochem 57:871–881, 2009)     

MUC1 and cancer

The MUC1 membrane mucin was first identified as the molecule recognised by mouse monoclonal antibodies directed to epithelial cells, and the cancers which develop from them. Cloning the gene showed that the extracellular domain is made up of highly conserved repeats of 20 amino acids, the actual number varying between 25 and 100 depending on the allele. Each tandem repeat contains five potential glycosylation sites, and between doublets of threonines and serines lies an immunodominant region which contains the epitopes recognised by most of the mouse monoclonal antibodies. The O-glycans added to the mucin produced by the normal breast are core 2 based and can be complex, while the O-glycans added to the breast cancer mucin are mainly core 1 based. This means that some core protein epitopes in the tandem repeat which are masked in the normal mucin are exposed in the cancer associated mucin. Since novel carbohydrate epitopes are also carried on the breast cancer mucin, the molecule is antigenically distinct from the normal breast mucin. (Changes in glycosylation in other epithelial cancers have been observed but are not so well documented.) Immune responses to MUC1 have been seen in breast and ovarian cancer patients and clinical studies have been initiated to evaluate the use of antibodies to MUC1 and of immunogens based on MUC1 for immunotherapy of these patients. The role of the carbohydrates in the immune response and in other interactions with the effector cells of the immune system is of particular interest and is discussed.     

Expression of carbohydrate epitopes L2/HNK-1 and L3 in the larva and imago of Drosophila melanogaster and Calliphora vicina

Summary The carbohydrate epitopes L2/HNK-1 and L3 belong to two overlapping families of adhesion molecules in the vertebrate, and probably the invertebrate nervous systems. To investigate their pattern of expression during the development of insects, cryosections of late third instar larvae and imagoes of Drosophila melanogaster and Calliphora vicina were studied by indirect immunofluorescence using several monoclonal antibodies to the L2/HNK-1 and one monoclonal antibody to the L3 epitope. Each monoclonal antibody to the L2/HNK-1 epitope showed a different immunohistological staining pattern, which differed from that of the L3 monoclonal antibody. In both insect species the immunohistological staining patterns for the two carbohydrate epitopes were similar at the two developmental stages, with immunoreactivity not confined to the nervous system. In larvae, immunoreactivities of the monoclonal antibodies L2.334 and L3.492 were predominantly associated with the extracellular matrix as indicated by co-localization with laminin, particularly in the imaginal discs, while L2.349 revealed a more cell surface-associated distribution. In imagoes, immunoreactivities were detectable in most organs studied.     

Alkali-catalyzed β-elimination of periodate-oxidized glycans: A novel method of chemical deglycosylation of mucin gene products in paraffin embedded sections

Altered expression of mucin gene products has been described in many epithelial cancers including colorectal cancer. However, mucins are heavily O-glycosylated making the study of apomucin expression difficult. In this study, we describe a novel method of chemical deglycosylation of mucin gene products on paraffin embedded formalin-fixed tissue sections. In the normal and cancerous colorectum, our results suggest that alkali-catalyzed -elimination of periodate oxidized glycan method of chemical deglycosylation modifies the structure of carbohydrates sensitive to mild periodate oxidation resulting in less steric hindrance and selectively removes Tn and sialyl-Tn structures, partially exposing the underlying apomucin epitopes. Using this method, we have demonstrated that the MUC1 tandem repeat epitope recognized by MAb 139H2 is masked predominantly due to steric hindrance by carbohydrate structures whereas the MUC2 tandem repeat epitope recognized by MAb CCP58 and pAb MRP and the MUC3 tandem repeat epitope recognized by pAb M3P are masked by the presence of carbohydrate side chains O-linked to Ser/Thr residues within the epitope. Considerable differences in the level and pattern of expression of the epitopes in the tandem repeat region of apomucins of MUC1, MUC2, and MUC3 were observed between normal and cancerous colorectal cancer tissues. We conclude that this novel chemical deglycosylation method that causes selective cleavage of distinct glycans will be useful in unmasking various mucin gene products and glycoproteins containing similar O-glycosidic linkages in the tissue sections of formalin-fixed paraffin embedded normal and pathological tissues.     

Anti-(human LFA-1) monoclonal antibodies bind P815 murine tumour cells

Summary Using anti-CD11a and anti-CD18 monoclonal antibodies (mAbs) directed respectively against the and the chains of LFA-1, we obtained an important and specific staining of P815 murine tumour cells. Both ascitic and cultured cells displayed a positive staining. Other murine tumours of haematopoietic origin, as well as lymphocytes or lymphoblasts from DBA/2 mice, were not labelled by the same monoclonal antibodies. These results were surprising since, to our knowledge, no case of cross-reaction between species has been reported with LFA-1. Moreover, competition assays showed that epitopes recognized by the two anti-CD11a antibodies were different from those identified by H35.89.9, a mAb raised against the murine LFA-1 chain. Using allogeneic cytotoxic T lymphocytes, we also showed that anti-(human LFA-1) mAbs were unable to block the lysis of P815 by these effector cells. Thus, the putative functional properties of these structures, as well as their importance from an antigeneic point of view, remain to be assessed.     

Production and characterization of human anti-V3 monoclonal antibodies from the cells of HIV-1 infected Indian donors

ABSTRACT: BACKGROUND: Analysis of human monoclonal antibodies (mAbs) developed from HIV-1 infected donors have enormously contributed to the identification of neutralization sensitive epitopes on the HIV-1 envelope glycoprotein. The third variable region (V3) is a crucial target on gp120, primarily due to its involvement in co-receptor (CXCR4 or CCR5) binding and presence of epitopes recognized by broadly neutralizing antibodies. METHODS: Thirty-three HIV-1 seropositive drug naive patients (18 males and 15 females) within the age range of 20--57 years (median = 33 years) were recruited in this study for mAb production. The mAbs were selected from EBV transformed cultures with conformationally constrained Cholera-toxin-B containing V3C (V3C-CTB) fusion protein. We tested the mAbs for their binding with HIV-1 derived proteins and peptides by ELISA and for neutralization against HIV-1 viruses by TZM-bl assays. RESULTS: We isolated three anti-V3 mAbs, 277, 903 and 904 from the cells of different individuals. The ELISA binding revealed a subtype-C and subtype-A specific binding of antibody 277 and 903 while mAb 904 exhibited cross reactivity also with subtype-B V3. Epitope mapping of mAbs with overlapping V3 peptides showed exclusive binding to V3 crown. The antibodies displayed high and low neutralizing activity against 2/5 tier 1 and 1/6 tier 2 viruses respectively. Overall, we observed a resistance of the tier 2 viruses to neutralization by the anti-V3 mAbs, despite the exposure of the epitopes recognized by these antibodies on two representative native viruses (Du156.12 and JRFL), suggesting that the affinity of mAb might equally be crucial for neutralization, as the epitope recognition. CONCLUSIONS: Our study suggests that the anti-V3 antibodies derived from subtype-C infected Indian patients display neutralization potential against tier 1 viruses while such activity may be limited against more resistant tier 2 viruses. Defining the fine epitope specificities of these mAbs and further experimental manipulations will be helpful in identification of epitopes, unique to clade C or shared with non-clade C viruses, in context of V3 region.     

A Comprehensive Study of Neutralizing Antigenic Sites on the Hepatitis E Virus (HEV) Capsid by Constructing,Clustering, and Characterizing a Tool Box

The hepatitis E virus (HEV) ORF2 encodes a single structural capsid protein. The E2s domain (amino acids 459–606) of the capsid protein has been identified as the major immune target. All identified neutralizing epitopes are located on this domain; however, a comprehensive characterization of antigenic sites on the domain is lacking due to its high degree of conformation dependence. Here, we used the statistical software SPSS to analyze cELISA (competitive ELISA) data to classify monoclonal antibodies (mAbs), which recognized conformational epitopes on E2s domain. Using this novel analysis method, we identified various conformational mAbs that recognized the E2s domain. These mAbs were distributed into 6 independent groups, suggesting the presence of at least 6 epitopes. Twelve representative mAbs covering the six groups were selected as a tool box to further map functional antigenic sites on the E2s domain. By combining functional and location information of the 12 representative mAbs, this study provided a complete picture of potential neutralizing epitope regions and immune-dominant determinants on E2s domain. One epitope region is located on top of the E2s domain close to the monomer interface; the other is located on the monomer side of the E2s dimer around the groove zone. Besides, two non-neutralizing epitopes were also identified on E2s domain that did not stimulate neutralizing antibodies. Our results help further the understanding of protective mechanisms induced by the HEV vaccine. Furthermore, the tool box with 12 representative mAbs will be useful for studying the HEV infection process.