Species-specificity of monoclonal antibodies recognising Prevotella intermedia and Prevotella nigrescens

Abstract Prevotella intermedia and Prevotella nigrescens are not easily distinguished, making it difficult to assess their roles in disease. This study examined the specificity of three monoclonal antibodies (mAbs) for these species. Differentiation between P. intermedia (13 isolates) and P. nigrescens (24 isolates) was by the electrophoretic mobility of their malate and glutamate dehydrogenase enzymes or by DNA homology grouping. All P. intermedia reacted strongly with mAb 40BI3.2.2 whereas P. nigrescens strains did not. Monoclonal antibodies 37BI6.1 and 39BI1.1.2 recognised all strains of both species but most P. nigrescens reacted weakly with mAb 39BI1.1.2. Monoclonal antibody 40BI3.2.2 therefore recognises an antigen specific for P. intermedia but not P. nigrescens and provides an easy and reliable means of distinguishing between these species. Three vaginal isolates identified biochemically as P. intermedia had enzymes with mobilities corresponding to neither P. intermedia nor P. nigrescens . These isolates were not recognised by mAbs 39BI1.1.2 or 40BI3.2.2 and may represent an undescribed taxon within this group of organisms.     

Epitope analyses of pneumococcal surface protein A: a combination of two monoclonal antibodies detects 94% of clinical isolates

Immunisation of BALB/c mice with seven heat-treated Norwegian clinical isolates of Streptococcus pneumoniae of different serotypes elicited mainly monoclonal antibodies (mAbs) to pneumococcal surface protein A (PspA). It was remarkable that the fusions resulted only in a few mAbs directed against other protein antigens. Dot blot analysis with 16 mAbs using clinical isolates representing 23 different capsular types and the uncapsulated reference strain R36A showed that some of the mAbs bound to PspA epitopes expressed by a low number of strains whereas others bound to broadly distributed epitopes. On the basis of their reactivities, seven of these mAbs could be divided into two groups recognising different subsets of pneumococci. The three mAbs in the narrow reacting group bound to epitopes found in 21-25% of the strains whereas the four mAbs in the broad reacting group detected more than 57% of the analysed strains. The epitopes for these seven antibodies were surface exposed on live exponential phase grown pneumococci as shown by flow cytometry. The finding that a combination of mAb 180,C-1 (IgG2a) from the first group and mAb 170,E-11 (IgG2a) from the second group detected 94% of the examined strains is interesting because PspA has been reported by others to be a serological highly variable protein.     

Ruthenium red staining reveals surface fibrils and a layer external to the cell wall in Streptococcus salivarius HB and adhesion deficient mutants

Ruthenium red staining revealed both the long and short classes of cell surface fibril in thin sections of Streptococcus salivarius HB, indicating that the fibrils contained polyanionic polymers, probably polysaccharides. Also visible was a 16.2 +/- 2.2 nm thick ruthenium red staining layer (RRL) outside the 16.7 +/- 2.2 nm thick cell wall. The fibrils could not be seen after conventional glutaraldehyde and osmium fixation. The RRL was protease resistant and was not involved in septum formation. Loss of the fibrils after protease treatment coincided with a decrease of 54% in cell surface hydrophobicity, indicating that cell surface hydrophobicity was due partly to fibrils and partly to the RRL. There was no correlation between the lengths of fibrils as measured on whole cells after negative staining and on thin sections of ruthenium red stained cells. The thickness of the RRL was the same in three adhesion deficient mutants--strains HB-7, HB-V5 and HB-V51--with various fibril lengths. However, a completely bald mutant, HB-B, had a significantly thicker RRL than S. salivarius HB, although it was unable to adhere to buccal epithelial cells, and it could not co-aggregate with Veillonella parvula V1. The RRL therefore did not contain adhesins.     

Production and characterisation of monoclonal antibodies to salmon pancreas disease virus.

Six mouse monoclonal antibodies (mAbs) specific to salmon pancreas disease virus (SPDV) were produced following immunisation with purified virus preparations. These mAbs and 2 mAbs resulting from an earlier investigation were characterised. None of the mAbs possessed virus neutralising activity but all reacted with 4 geographically different SPDV isolates as determined by indirect immunofluorescence. Three mAbs produced positive immunostaining with Western blots of SPDV proteins. The 4H1 mAb reacted with the 53 kDa structural E1 glycoprotein present in virus-infected cells and in gradient-purified virus. Two mAbs, 5A5 and 7B2, which exhibited unusual immunofluorescence staining of the nuclear margin, reacted with a 35 kDa protein, which is present in gradient-purified virus and which is considered to be the capsid protein. A sandwich ELISA, based on the use of mAb 2D9 for capture and a biotinylated conjugate of mAb 7A2 for detection, detected SPDV antigen in virus-infected Chinook salmon embryo-214 cells and gradient-purified virus. These mAbs may be of use in pathogenesis studies and in diagnostic test development.     

Epitope mapping of pneumococcal surface protein A of strain Rx1 using monoclonal antibodies and molecular structure modelling

Pneumococcal surface protein A (PspA) is an antigenic variable vaccine candidate of Streptococcus pneumoniae. Epitope similarities between PspA from the American vaccine candidate strain Rx1 and Norwegian clinical isolates were studied using PspA specific monoclonal antibodies (mAbs) made against clinical Norwegian strains. Using recombinant PspA/Rx1 fragments and immunoblotting the epitopes for mAbs were mapped to two regions of amino acids, 1-67 and 67-236. The discovered epitopes were visualized by modelling of the PspA:Fab part of mAb in three dimensions. Flow cytometric analysis showed that the epitopes for majority of mAbs were accessible for antibody binding on live pneumococci. Also, the epitopes for majority of the mAbs are widely expressed among clinical Norwegian isolates.     

Protective activity of monoclonal antibodies to genome-derived neisserial antigen 1870, a Neisseria meningitidis candidate vaccine

Genome-derived neisserial Ag (GNA) 1870 is a meningococcal vaccine candidate that can be subdivided into three variants based on amino acid sequence variability. Variant group 1 accounts for approximately 60% of disease-producing group B isolates. The Ag went unrecognized until its discovery by genome mining because it is expressed in low copy number by most strains. To investigate the relationship between Ab binding to GNA1870 and complement-mediated protective functions, we prepared a panel of four murine IgG mAbs against rGNA1870 (variant 1) and evaluated their activity against nine genetically diverse encapsulated Neisseria meningitidis strains expressing subvariants of variant 1 GNA1870. Based on flow cytometry with live encapsulated bacteria, surface accessibility of the epitopes recognized by the mAbs appeared to be low in most strains. Yet mAb concentrations <1 to 5 micro g/ml were sufficient to elicit bactericidal activity with human complement and/or activate C3b deposition on the bacterial surface. Certain combinations of mAbs were highly bactericidal against strains that were resistant to bactericidal activity of the respective individual mAbs. The mAbs conferred passive protection against bacteremia in infant rats challenged by strains resistant to bacteriolysis, and the protective activity paralleled the ability of the mAb to activate C3b deposition. Thus, despite low GNA1870 surface exposure, anti-GNA1870 variant 1 Abs are bactericidal and/or elicit C3b deposition and confer protection against bacteremia caused by encapsulated N. meningitidis strains expressing GNA1870 subvariant 1 proteins. The data support GNA1870 as a promising vaccine candidate for prevention of meningococcal group B disease caused by GNA1870 variant 1 strains.     

Monoclonal antibodies that recognize calcium-dependent structures of human thrombospondin. Characterization and mapping of their epitopes

Monoclonal antibodies (mAbs) raised against reduced and alkylated thrombospondin (TSP) were screened for the ability to react with Ca2+-replete TSP versus EDTA-treated TSP. Two mAbs designated A6.1 and D4.6 were found to react much more strongly with TSP after EDTA treatment. The dissociation constants for these mAbs were measured in 5 mM EDTA and found to be 6 X 10(-10) M for A6.1 and 7 X 10(-9) M for D4.6. Binding to A6.1 was undetectable in the presence of 1 mM Ca2+ while binding of D4.6 occurred with about 100-fold lower affinity. The Ca2+ concentration dependence of A6.1 binding was broad with a midpoint near 50 microM free Ca2+ while that of D4.6 showed a sharp transition below 0.1 microM. Upon dialysis of EDTA-treated TSP into Ca2+ containing buffer, the binding of the mAbs was prevented or decreased, indicating reversibility of the conformational transition induced by the initial removal of Ca2+ . Mg2+ can compete with the Ca2+ binding sites involved in mAb binding, but TSP dialyzed from Ca2+ into Mg2+ binds the two mAbs as well as EDTA-treated TSP, indicating that Mg2+ cannot maintain the Ca2+-replete structure of TSP. The proteolytic fragments of TSP with which the two mAbs react were determined by probing Western blots of digests of TSP with the mAbs. A6.1 reacts with the 70-kDa fragment generated by chymotrypsin in EDTA which contains the interchain disulfide bonds of TSP and the binding site(s) for type V collagen (Mumby, S. M., Raugi, G. J., and Bornstein, P. (1984) J. Cell Biol. 98, 646-652). D4.6 reacts with fragments of 140 and 120 kDa found in digests of Ca2+-replete TSP which are absent from digests in EDTA. Electron microscopy of rotary shadowed, carbon-coated replicas of TSP mAb complexes confirms the Ca2+ sensitivity of mAb binding and has been used to localize the epitopes for both mAbs on the three-dimensional structure of TSP.     

Destructive effects of murine arthritogenic antibodies to type II collagen on cartilage explants <Emphasis Type="Italic">in vitro</Emphasis>

Certain monoclonal antibodies (mAbs) to type II collagen (CII) induce arthritis in vivo after passive transfer and have adverse effects on chondrocyte cultures and inhibit self assembly of collagen fibrils in vitro. We have examined whether such mAbs have detrimental effects on pre-existing cartilage. Bovine cartilage explants were cultured over 21 days in the presence of two arthritogenic mAbs to CII (CIIC1 or M2139), a non-arthritogenic mAb to CII (CIIF4) or a control mAb (GAD6). Penetration of cartilage by mAb was determined by immunofluorescence on frozen sections and correlated with changes to the extracellular matrix and chondrocytes by morphometric analysis of sections stained with toluidine blue. The effects of mAbs on matrix components were examined by Fourier transform infrared microspectroscopy (FTIRM). A possible role of Fc-binding was investigated using F(ab)₂ from CIIC1. All three mAbs to CII penetrated the cartilage explants and CIIC1 and M2139, but not CIIF4, had adverse effects that included proteoglycan loss correlating with mAb penetration, the later development in cultures of an abnormal superficial cellular layer, and an increased proportion of empty chondrons. FTIRM showed depletion and denaturation of CII at the explant surface in the presence of CIIC1 or M2139, which paralleled proteoglycan loss. The effects of F(ab)₂ were greater than those of intact CIIC1. Our results indicate that mAbs to CII can adversely affect preformed cartilage, and that the specific epitope on CII recognised by the mAb determines both arthritogenicity in vivo and adverse effects in vitro. We conclude that antibodies to CII can have pathogenic effects that are independent of inflammatory mediators or Fc-binding.     

Strain-related differences in antibody-mediated changes in gene expression are associated with differences in capsule and location of binding

We recently established that antibody (Ab)-binding can induce gene expression changes in a serotype A strain (H99) of the pathogenic yeast, Cryptococcus neoformans. That study showed that monoclonal antibodies (mAbs) differing in epitope specificity and protective efficacy elicited differences in gene expression. Because many mAbs bind to serotypes A and D strains differently, we now investigate the binding of one mAb to two strains representing these serotypes. Cells of the serotype A strain H99 and the serotype D strain 24067 were incubated with near saturating concentrations of the IgG1 capsule-binding mAb 18B7 or MOPC, an irrelevant mAb matched control. Comparative immunofluorescence analysis of mAb 18B7 binding revealed that it bound closer to the cell wall in H99 than 24067, where it was associated with decreased or increased cell diameter, respectively. A comparison of encapsulated cell compressibility showed that strain 24067 was more compressible than that of strain H99. RNA was extracted and used for gene expression analysis using the C. neoformans JEC21 genomic microarray. After 1h incubation with mAb 18B7, there were just 2 gene expression changes observed with strain 24067 or strain JEC21, unlike the 43 seen with strain H99. After 4h incubation with mAb 18B7, there were 14 and 140 gene expression changes observed with strain 24067 and JEC21, respectively. Thus, C. neoformans strains differ both in the response and the time of response to mAb binding and these differences may reflect differences in the location of Ab binding, Ab-mediated changes in cell diameter and compressibility of the capsular polysaccharide.     

Production of Monoclonal Antibodies to Barley Mild Mosaic Virus and Their Use for Strain Differentiation

A panel of five stable hybridoma cell lines secreting mono- clonal antibodies (mAbs) were produced using a French mechanically transmitted isolate of barley mild mosaic virus (BaMMV-MF) as antigen. All mAbs reacted with BaMMV-MF in two enzyme-linked immunosorbent assay (ELISA) formats: triple antibody sandwich (TAS)-ELISA and antigen-coated plate (ACP)-ELISA. These mAbs recognized epitopes, present on both degraded virions and intact particles. Four mAbs (5C8, 1D5, 1B12, 1A12) belong to the immunoglobulin (Ig)G class and one mAb (3A9) represents an IgM. The five mAbs were compared in TAS- and ACP-ELISA for reactivity with numerous French isolates. These isolates were detected in TAS- and ACP-ELISA with four mAbs (5C8, 1D5, 1B12, 3A9). In both ELISA systems the mAb 1A12 recognized only an epitope specific for BaMMV-MF. All mAbs, except 1A12 recognized also the German (BaMMV-MG), Italian (BaMMV-I) and Japanese (BaMMV-Ka1) isolates in both TAS- and ACP-ELISA. The Japanese isolate (BaMMV-Na1) only reacted with two mAbs (1D5, 5C8) in TAS-ELISA. Only one mAb (3A9) reacted with BaMMV-MF, BaMMV-PF, BaMMV-I,BaMMV-MG and BaMMV-Ka1 in Western blot. These mAbs make it possible to distingish between the three BaMMV serotypes.     

Characterization of monoclonal antibodies that recognize common epitopes located on O antigen of lipopolysaccharide of serotypes 1, 9 and 11 of Actinobacillus pleuropneumoniae

Abstract Seven murine monoclonal antibodies (mAbs) against serotype 1 of Actinobacillus (Haemophilus) pleuropneumoniae (reference strain Shope 4074) were produced and characterized. All hybridomas secreting mAbs were reactive with whole-cell antigens from reference strains of serotypes 1, 9 and 11, except for mAb 5D6 that failed to recognize serotype 9. They did not react with other taxonomically related Gram-negative organisms tested. The predominant isotype was immunoglobulin (Ig) M, although IgG_2a, IgG_2b, and IgG₃ were also obtained. The epitopes identified by these mAbs were resistant to proteinase K treatment and boiling in the presence of sodium dodecyl sulfate and reducing conditions; however, they were sensitive to sodium periodate treatment. Enhanced chemiluminescence-immunodetection assay showed that mAbs could be divided in two groups according to the patterns of immunoreaction observed. Group I (mAbs 3E10, 4B7, 9H5 and 11C3) recognized a ladder-like banding profile consistent with the O antigen of lipopolysaccharide (LPS) from smooth strains. Group II (mAbs 3B10 and 9H1) recognized a long smear of high molecular weight which ranged from 60 to 200 kDa. The mAbs were tested against 96 field isolates belonging to serotypes 1, 5, 9, 11 and 12, which had previously been classified by a combination of serological techniques based on polyclonal rabbit sera (counterimmunoelectrophoresis, immunodiffusion and coagglutination). The panel of mAbs identified all isolates of serotypes 9 and 11, but only 66% of those belonging to serotype 1. This may suggest the existence of antigenic heterogeneity among isolates of A. pleuropneumoniae serotype 1. These mAbs reacted with epitopes common to serotypes 1, 9 and 11 of Actinobacillus pleuropneumoniae which were located on the O antigen of LPS.     

Localization of conserved B-cell epitopes among encapsulated and non-encapsulated Haemophilus influenzae P2 porin proteins using synthetic peptides.

The P2 outer membrane protein of Haemophilus influenzae belongs to a class of apparently ubiquitous proteins in Gram-negative bacteria that function as porins. Murine hybridomas raised to the P2 protein and synthetic peptides were used to investigate the structural and antigenic relationships among P2 proteins of encapsulated and non-encapsulated H. influenzae. Three monoclonal antibodies (mAbs), P2-17, P2-18 and P2-19, recognizing epitopes on the P2 protein, as shown by Western immunoblotting of outer membrane preparations, and purified and recombinant P2 proteins are described. The epitopes reactive with the mAbs were widely distributed among H. influenzae strains since 70-100% of strains of encapsulated and non-encapsulated isolates collected worldwide were recognized by individual mAbs. None of the mAbs reacted with H. parainfluenzae or other bacterial species. The peptide composition of P2 epitopes was determined by analysis of mAb reactivity with a series of overlapping synthetic peptides that covered the amino acid sequences of H. influenzae type b. The domains recognized by these mAbs were completely distinct. mAb P2-18, reactive with an epitope conserved among all H. influenzae P2 porin molecules which were screened, recognized a peptide corresponding to the N-terminal segment (residues 1-14). The P2-17- and P2-19-specific epitopes were located between residues 28 and 55, and 101 and 129, respectively. None of the epitopes were exposed on the cell surface since no mAbs bound to intact live bacteria.(ABSTRACT TRUNCATED AT 250 WORDS)     

Monoclonal antibodies as probes for detecting lipopolysaccharide expression on Escherichia coli from different growth conditions.

Monoclonal antibody (mAb) probes were used to investigate the expression of lipopolysaccharide (LPS) on four Escherichia coli strains, grown under a variety of conditions in batch culture which mimicked some of the in vivo environmental conditions of an infected host. Techniques of silver staining, immunoblotting, whole cell ELISA and flow cytometry were all used to monitor the expression of LPS on the bacteria and the binding of the anti-LPS mAbs. Growth in heat-inactivated sheep serum and magnesium-depleted conditions demonstrated increased expression of LPS core and subsequent increased binding of anti-core mAbs. Magnesium-depleted conditions also resulted in decreased production of O-polysaccharide material. Iron-depleted bacteria showed only minor changes in LPS expression, although increased binding of anti-core mAbs was observed. Nitrogen-deficient/high-carbon conditions, chosen to promote capsule production, resulted in increased expression of O-polysaccharide and decreased binding of anti-core mAbs.     

Characterization of monoclonal antibodies to O-antigen of lipopolysaccharide of Actinobacillus pleuropneumoniae serotype 2 and their use in the classification of field isolates

Abstract Monoclonal antibodies (mAbs) against Actinobacillus pleuropneumoniae serotype 2 (reference strain Shope 4226 and field isolate F46) were produced. Twelve hybridoma clones were selected against both strains, and all the antibodies secreted were found to be reactive with whole-cell antigen of the homologous strain in ELISA, whereas only one mAb was reactive in slide agglutination test. The predominant antibody classes were IgG2b and IgG3, although IgG1 and IgM were also obtained. Immunoblot assay showed that mAbs could recognize a ladder band profile which is in accordance with the O-antigen of lipopolysaccharide. Most of the epitopes involved were resistant to proteinase K and also to boiling in the presence of sodium dodecyl sulfate and reducing conditions, but they were sensitive to periodic acid. The 12 mAbs recognized neither reference strains of the remaining A. pleuropneumoniae serotypes nor other taxonomically related Gram-negative organisms. The suitability of mAbs for serotyping of field isolates was also examined, and a high correlation (97.4%) was found between the results previously established by indirect hemagglutination with polyclonal rabbit sera and those obtained by ELISA with mAbs. The panel of mAbs described in this study was found to be extremely useful for identifying field isolates belonging to serotype 2 and could be used as a complementary serotyping method.     

Generation of monoclonal antibodies to Macaca mulatta (rhesus) IgA

One IgG1 and five IgM murine monoclonal antibodies (mAb) specific for rhesus (Rh) IgA were generated. These mAbs bound to Rh IgA but not IgG or IgM when tested by enzyme-linked immunosorbent assay. Immunoblotting revealed that the mAbs reacted with the alpha heavy chain of Rh but not human IgA. The IgG1 anti-Rh IgA mAb detected IgA-producing cells in sections of monkey gut examined by immunofluorescent staining. These mAbs should be useful for characterizing IgA responses in the Rh monkey.     

Physicochemical and structural investigation of the surfaces of some anaerobic subgingival bacteria.

The surfaces of nine clinical isolates of Porphyromonas gingivalis, Prevotella intermedia, Actinobacillus actinomycetemcomitans, and Peptostreptococcus micros and that of laboratory strain P. gingivalis W83 were studied by using contact angle measurements, X-ray photoelectron spectroscopy, infrared spectroscopy, microelectrophoresis of whole cells, and transmission electron microscopy of whole and sectioned cells. P. intermedia strains were hydrophilic, as judged from their small water contact angles, and had highly negative zeta potentials, consistent with the presence of a prominent ruthenium red (RR)-staining layer and fibrillar appendages which are probably partly carbohydrate. The two clinical isolates of P. gingivalis were also hydrophilic and highly negatively charged despite the presence of prominent fibrils, which usually yield less negative zeta potentials. This finding suggests that the RR-staining layer dominates the suspension characteristics of P. gingivalis and P. intermedia strains. P. gingivalis W83 had no demonstrable fibrils and a morphologically distinct RR-staining layer, and it was more hydrophobic than the two clinical isolates of P. gingivalis. P. micros isolates were hydrophobic and much less negatively charged than the other species. The A. actinomycetemcomitans strains displayed long, prominent fibrils and a very thin RR-staining layer, which resulted in high hydrophobicity but distinctly different zeta potentials for the two. Physicochemical data on microbial cell surfaces usually have clear and predictable relationships with each other. For the strains in this study that did not follow these relationships, their aberrant behavior could be explained as due to a masking effect caused by specific surface architecture. We conclude that this combined analysis provides a detailed image of subgingival bacterial surface architecture.     

Physicochemical and structural investigation of the surfaces of some anaerobic subgingival bacteria.

The surfaces of nine clinical isolates of Porphyromonas gingivalis, Prevotella intermedia, Actinobacillus actinomycetemcomitans, and Peptostreptococcus micros and that of laboratory strain P. gingivalis W83 were studied by using contact angle measurements, X-ray photoelectron spectroscopy, infrared spectroscopy, microelectrophoresis of whole cells, and transmission electron microscopy of whole and sectioned cells. P. intermedia strains were hydrophilic, as judged from their small water contact angles, and had highly negative zeta potentials, consistent with the presence of a prominent ruthenium red (RR)-staining layer and fibrillar appendages which are probably partly carbohydrate. The two clinical isolates of P. gingivalis were also hydrophilic and highly negatively charged despite the presence of prominent fibrils, which usually yield less negative zeta potentials. This finding suggests that the RR-staining layer dominates the suspension characteristics of P. gingivalis and P. intermedia strains. P. gingivalis W83 had no demonstrable fibrils and a morphologically distinct RR-staining layer, and it was more hydrophobic than the two clinical isolates of P. gingivalis. P. micros isolates were hydrophobic and much less negatively charged than the other species. The A. actinomycetemcomitans strains displayed long, prominent fibrils and a very thin RR-staining layer, which resulted in high hydrophobicity but distinctly different zeta potentials for the two. Physicochemical data on microbial cell surfaces usually have clear and predictable relationships with each other. For the strains in this study that did not follow these relationships, their aberrant behavior could be explained as due to a masking effect caused by specific surface architecture. We conclude that this combined analysis provides a detailed image of subgingival bacterial surface architecture.     

Surface structures, haemagglutination and cell surface hydrophobicity of Bacteroides fragilis strains.

Nineteen strains of Bacteroides fragilis were examined by negative staining for surface structures. One strain (ATCC 23745) possessed peritrichous fibrils, 16 strains carried peritrichous fimbriae and two strains carried no surface structures. The fimbriae had a diameter of 2.1 +/- 0.25 nm and appeared to be 'curly'. Only a small proportion (4 to 41%, depending on the strain) of cells in a population carried fimbriae or fibrils. Strain A312 Showed phase variation of fimbriae as expression of fimbriae was repressed at 20 degrees C and in early exponential phase at 37 degrees C. The fibrils on strain ATCC 23745 did not exhibit phase variation in response to changes in incubation temperature, growth phase or growth in two different media. Capsules were demonstrated by the Indian ink method on 18 of the 19 strains, varying in size from strain to strain and within the same population. Cultures often contained both capsulate and noncapsulate cells. All strains possessed an electron dense ruthenium red staining layer between 7.9 and 23.9 nm in width attached to the outer membrane. Cell surface hydrophobicity quantified by the hexadecane partition assay gave low values ranging from 6.6 to 52.1%. Only a few strains were able to haemagglutinate and these were only weakly active. There was no correlation between cell surface hydrophobicity, haemagglutinating activity and surface structures.     

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.     

Insertion elements in Staphylococcus intermedius

Staphylococcus intermedius cultures from dogs, pigeons, horses and mink were investigated for the prevalence of the insertion elements IS 256 and IS 257 in relation to their antibiotic resistance. Copies of IS 256 could not be detected in any of the Staph. intermedius isolates tested whereas single copies of IS 257 occurred in the isolates from dogs and horses. The mink strains did not harbour IS 257 elements, whereas Staph. intermedius isolates from pigeons carried multiple copies of IS 257 as predicted from the hybridization patterns obtained with a gene probe derived from the internal part of the IS 257 -encoded transposase gene. Independently of the origin of the Staph. intermedius isolates, all IS 257 copies were found to be located in the chromosomal DNA. The large number of chromosomal IS 257 copies in the pigeon strains might help to explain chromosomal multiresistance in many of those strains.