Sodium Metaperiodate Treatment Research Articles

Polyclonal antibodies to Paracoccidioides brasiliensis are able to recognise antigens from different strains from Paracoccidioides species complex, including Paracoccidioides lutzii LDR2.

Since the new species Paracoccidioides lutzii emerged in 2009, much has been researched on strains previously considered atypical. Still, there is no consensus about recognition of antigens from P.lutzii by antibodies directed to other Paracoccidioides species, which can have great impact on Paracoccidioidomycosis (PCM) diagnosis. Current research investigated soluble protein/carbohydrate epitopes from P.lutzii LDR2, Paracoccidioidesrestrepiensis B339 and Paracoccidioidesamericana LDR3 recognised by IgG directed to Paracoccidioidesbrasiliensis. Cell free antigens (CFA) were analysed by: (a)silver and periodic acid-Schiff staining of SDS-PAGE; (b)immunoblot (IB) with rabbit IgG anti-P.brasiliensis Pb18; (c)IB and ELISA with a pool of PCM patients' sera before and after treatment with sodium metaperiodate (SMP) to oxidise carbohydrate epitopes. Both rabbit and human immune sera recognised several antigens of P.lutzii LDR2, P.restrepiensis B339 and P.americana LDR3. P.lutzii's gp43 was not observed in IB or silver/PAS staining. SMP treatment affected reactions with all 3 CFAs, but more intensely with antigens from P.lutzii LDR2. In conclusion, antibodies directed to P.brasiliensis recognised antigens from P.lutzii LDR2. The use of any of the recognised antigens in a broad spectrum diagnostic model for Paracoccidioides species complex needs to be further investigated.

Metaperiodate deglycosylation of Strongyloides venezuelensis larvae: Immunochemical characterization and antigen production for human strongyloidiasis diagnosis

Strongyloidiasis is an important helminthiasis affecting million people worldwide. The aim of this study was to use sodium metaperiodate (MP) treatment to immunochemically characterize Strongyloides venezuelensis filariform larvae and use MP-treated heterologous antigen to detect IgG and subclasses in serum. Samples from individuals with definitive diagnosis of strongyloidiasis (n = 50), other parasitic diseases (n = 60) and negative endemic (n = 50) were tested. TG-ROC and two-way ANOVA were applied. MP-treatment resulted on differential localization of carbohydrates at larval structure and no carbohydrate content in saline extract (SE). Electrophoretic profiles were similar before and after treatment. ELISA sensitivity and specificity were: 90%; 88.2% for SE and 92.0%; 94.6% for MP, respectively. When using MP treated antigen we observed reduction in IgG1 and IgG3 detection in strongyloidiasis group and decrease of cross reactions in control groups. Our data demonstrate the role of carbohydrate residues in cross reactions and on the recognition of anti-Strongyloides IgG and its subclasses.

A 53 KDa Glycan Antigen of Hydatid Cyst Wall May Involve in Evasion from Host Immune System

Background:Recent studies have shown that similar host glycan antigens are expressed by helminths such as Echinococcus granulosus hydatid cysts to evade from host immune system. In this work to investigate these antigens further, immunological cross-reactivity between human sera and hydatid cyst wall antigens has been investigated.Materials and Methods:Hydatid cyst wall antigens were used in enzyme-linked immunosorbent assay and Western immunoblotting and probed with pooled sera of hydatidosis patients and healthy controls. Sodium metaperiodate treatment was used to investigate glycan antigens.Results:A band with molecular weight about 53 KDa reacted with both hydatid patients' sera and also normal human sera. It has been shown that this band was a glycan antigen.Conclusions:A 53 KDa glycan antigen of hydatid cyst wall that reacted with all human sera may have an important role for evasion from host immune system.

Toxoplasma gondii tachyzoite-extract acts as a potent immunomodulator against allergic sensitization and airway inflammation

Epidemiological and experimental studies have shown an inverse relationship between infections with certain parasites and a reduced incidence of allergic diseases. We and others have shown that infection with Toxoplasma gondii prevents the development of allergy in mice. To establish whether this beneficial effect could be recapitulated by soluble products of this parasite, we tested an extract derived from T. gondii tachyzoites. Immunization of BALB/c mice with tachyzoites lysate antigen (TLA) elicited mixed Th1/Th2 responses. When TLA was applied together with the sensitizing ovalbumin (OVA), the development of allergic airway inflammation was reduced, with decreased airway hyperresponsiveness associated with reduced peribronchial and perivascular cellular infiltration, reduced production of OVA-specific Th2 cytokines in lungs and spleens and reduced levels of serum OVA-specific IgG1 as well as IgE-dependent basophil degranulation. Of note, TLA retained its immunomodulatory properties, inducing high levels of IL-6, TNFα, IL-10 and IL-12p70 in bone marrow-derived dendritic cells after heat-inactivation or proteinase K-treatment for disruption of proteins, but not after sodium metaperiodate-treatment that degrades carbohydrate structures, suggesting that carbohydrates may play a role in immunomodulatory properties of TLA. Here we show that extracts derived from parasites may replicate the benefits of parasitic infection, offering new therapies for immune-mediated disorders.

Antibiotic susceptibility of planktonic- and biofilm-grown staphylococci isolated from implant-associated infections: should MBEC and nature of biofilm formation replace MIC?

The purpose of this study was to develop an alternative, more clinically relevant approach to susceptibility reporting for implant-associated infections. Using 20 staphylococcal isolates, isolated from clinical implant infections, the majority (85 %) demonstrated biofilm-forming capabilities. A significantly increased minimum biofilm eradication concentration (MBEC) compared to minimum inhibitory concentration (MIC) breakpoint was obtained, with MBEC values greater than 256 µg ml-1 for the majority of bacteria. Such a vast increase was also demonstrated for isolates defined as negligible biofilm formers via crystal violet staining, likely due to the high protein content of biofilms, as confirmed by proteinase-K treatment. This study employed a variety of techniques to assess MIC and MBEC of the isolates tested. In addition, the nature of bacterial biofilm across a range of clinical isolates was investigated using crystal violet staining, sodium metaperiodate and proteinase-K treatment, and PCR analysis.Results/Key findings. Infection of medical implants is associated with increased rates of infection and increased bacterial tolerance to antibiotic strategies. Clinical significance is due to the presence of pathogens attached to biomaterial surfaces enclosed in an extracellular polymeric matrix termed the biofilm. This article highlights the importance of defining the clinical susceptibility of implant-associated infections in vitro using methods that are relevant to the biofilm phenotype in vivo, and highlights how current planktonic-based antimicrobial susceptibility tests are often misleading. The use of biofilm-relevant susceptibility tests would improve patient outcomes by enabling correct antimicrobial regimens to be rapidly identified, reducing treatment failure and halting the spread of antimicrobial-resistant strains.

Methicillin-Resistant Staphylococcus aureus Biofilms and Their Influence on Bacterial Adhesion and Cohesion.

Twenty-five methicillin-resistant Staphylococcus aureus (MRSA) isolates were characterized by staphylococcal protein A gene typing and the ability to form biofilms. The presence of exopolysaccharides, proteins, and extracellular DNA and RNA in biofilms was assessed by a dispersal assay. In addition, cell adhesion to surfaces and cell cohesion were evaluated using the packed-bead method and mechanical disruption, respectively. The predominant genotype was spa type t127 (22 out of 25 isolates); the majority of isolates were categorized as moderate biofilm producers. Twelve isolates displayed PIA-independent biofilm formation, while the remaining 13 isolates were PIA-dependent. Both groups showed strong dispersal in response to RNase and DNase digestion followed by proteinase K treatment. PIA-dependent biofilms showed variable dispersal after sodium metaperiodate treatment, whereas PIA-independent biofilms showed enhanced biofilm formation. There was no correlation between the extent of biofilm formation or biofilm components and the adhesion or cohesion abilities of the bacteria, but the efficiency of adherence to glass beads increased after biofilm depletion. In conclusion, nucleic acids and proteins formed the main components of the MRSA clone t127 biofilm matrix, and there seems to be an association between adhesion and cohesion in the biofilms tested.

Phenotypic and genotypic characterization of Staphylococci causing breast peri-implant infections in oncologic patients.

BackgroundStaphylococcus epidermidis and S. aureus have been identified as the most common bacteria responsible for sub-clinical and overt breast implant infections and their ability to form biofilm on the implant as been reported as the essential factor in the development of this type of infections. Biofilm formation is a complex process with the participation of several distinct molecules, whose relative importance in different clinical settings has not yet been fully elucidated. To our knowledge this is the first study aimed at characterizing isolates causing breast peri-implant infections.ResultsThirteen S. aureus and seven S. epidermidis causing breast peri-implant infections were studied.Using the broth microdilution method and the E-test, the majority of the strains were susceptible to all antibiotics tested. Methicillin resistance was detected in two S. epidermidis. All strains had different RAPD profiles and were able to produce biofilms in microtitre plate assays but, while all S. aureus carried and were able to express icaA and icaD genes, this was only true for one S. epidermidis. Biofilm development was glucose- and NaCl-induced (5 S. aureus and 1 S. epidermidis) or glucose-induced (the remaining strains). Proteinase K and sodium metaperiodate treatment had different effects on biofilms dispersion revealing that the strains studied were able to produce chemically different types of extracellular matrix mediating biofilm formation.All S. aureus strains harboured and expressed the atlA, clfA, FnA, eno and cna genes and the majority also carried and expressed the sasG (10/13), ebpS (10/13) genes.All S. epidermidis strains harboured and expressed the atlE, aae, embp genes, and the majority (six strains) also carried and expressed the fbe, aap genes.Genes for S. aureus capsular types 5 and 8 were almost equally distributed. The only leukotoxin genes detected were lukE/lukD (6/13).ConclusionsS. aureus and S. epidermidis breast peri-implant infections are caused by heterogeneous strains with different biofilm development mechanisms.Since the collagen adhesin (cna) gene is not ubiquitously distributed among S. aureus, this protein could have an important role in the cause of breast peri-implant infections.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-015-0368-x) contains supplementary material, which is available to authorized users.

The adhesion characteristics of 5 probiotic strains to the carp foregut mucus <I>in vitro</I>

Using immobilized mucosal model in vitro,combined with the isotope tracing method,the adhesion characteristics of Enterococci,Citrobacter isolated from carp intestines and Bacillus isolated from water to the carp foregut mucus were investigated. Results showed that after the modification with proteolytic enzymes or sodium metaperiodate,the relative adhesion percentages of all five bacteria were declined extremely significantly after they were treated with sodium metaperiodate(P0.01),but it was affected less by the proteolytic enzymes treatment(P0.05),indicating that main lectins existing on the cell surface of the five bacteria are glycoprotein compounds. After the modification with proteolytic enzymes or sodium metaperiodate of mucus,the relative adhesion percentages of part of five bacteria were declined significantly after the mucus was treated with proteolytic enzymes(P0.05),but it was affected significantly by the sodium metaperiodate treatment(P0.05),indicating that the special receptors of the microorganisms are protein compounds. According to the interactions such as exclusion,competition,and displacement,the five probiotic bacteria can reduce the relative adhesion percentages of part pathogens significantly. The inhibition of displacement to the pathogens was best,but the disparity was led by the different strain.

Assay of labile estrogen o-quinones, potent carcinogenic molecular species, by high performance liquid chromatography–electrospray ionization tandem mass spectrometry with phenazine derivatization

A sensitive and selective assay method for labile estrogen o-quinones, estrone (E 1)-2,3-quinone (Q), E 1-3,4-Q, estradiol (E 2)-2,3-Q and E 2-3,4-Q, based on the use of phenazine (Phz) derivatization with o-phenylenediamine and high performance liquid chromatography–electrospray ionization tandem mass spectrometry (HPLC–ESI-MS/MS) was described. The Phz derivatives of four estrogen o-quinones were purified by solid phase extraction and analyzed by HPLC–ESI-MS/MS. The protonated molecule was observed as a base peak for all Phz derivatives in their ESI-mass spectra (positive mode). In multiple reaction monitoring, the transition from [M+H] + to m/ z 231 was chosen for quantification. Calibration curves for the o-quinones were obtained using standard catechol estrogens after sodium metaperiodate treatment and Phz derivatization. Using this method, these four estrogen o-quinones were analyzed with the limit of quantification of 5 ng/ml in acetonitrile (MeCN)–blank matrix (1:4, v/v), respectively, on a basis of the weight of catechol estrogens. Assay accuracy and precision for four estrogen o-quinones were 89.6–113.0% and 3.1–12.6% (5, 125 and 2000 ng/ml in MeCN–blank matrix). Applications of this method enabled to determine the catalytic activities on hydroxylation and subsequent oxidation of E 1 and E 2 of Mushroom tyrosinase and rat liver microsomal fraction. It was confirmed by this method that tyrosinase exhibited 2- and 4-hydroxylation and further oxidation activities for catechols in the ring-A of estrogens. Whereas rat liver microsomal fraction possessed only 2- and 4-hydroxylation activities, and further oxidation activity for catechol estrogens was low.

Terminal α2-6 Linked Sialic Acid Expression On VWF Specifically Enhances Proteolysis by ADAMTS13.

Abstract 30VWF is a large plasma sialoglycoprotein that mediates platelet tethering at sites of vascular injury. VWF function is dependent upon VWF multimeric composition, which is regulated in plasma by ADAMTS13. ABO(H) blood group determinants expressed on VWF N-linked glycans significantly influence susceptibility to ADAMTS13 proteolysis. In this study, we investigated whether terminal sialic acid residues expressed on the N- and O-linked glycans of VWF may also regulate proteolysis by ADAMTS13. VWF was initially purified from human plasma (pdVWF) by cryoprecipitation and gel filtration. Subsequently, VWF sialylation was modified using specific exoglycosidases and quantified by lectin-binding ELISA. The rate of glycosidase-treated VWF proteolysis by ADAMTS13 was determined by incubation with recombinant ADAMTS13 and subsequent measurement of residual VWF collagen binding activity.Complete VWF deglycosylation has been shown to enhance the rate of proteolysis by ADAMTS13. In contrast, enzymatic desialylation of VWF by α2-3,6,8,9 neuraminidase (Neu-VWF) markedly impaired the rate of ADAMTS13-mediated VWF proteolysis. Neu-VWF collagen binding activity was reduced to only 50±14% by ADAMTS13, compared to 11±7% for untreated VWF (p<0.01) at the same time point. Despite this, Neu-VWF exhibited increased susceptibility to proteases other than ADAMTS13 (trypsin, chymotrypsin and cathepsin B; all p<0.05). VWF sialylation is therefore a specific enhancer of ADAMTS13-mediated proteolysis. Consequently, quantification and molecular distribution of VWF sialylation was examined by sequential digestion and HPLC analysis. Total sialic acid expression on pdVWF was 167nmol/mg, of which the majority (133.4nmol/mg or 80.1%) was present on its N-linked glycan chains. Interestingly, despite the resistance to ADAMTS13 proteolysis observed upon complete desialylation, digestion of pdVWF with α2-3 neuraminidase to remove predominantly O-linked sialic acid did not influence the rate of ADAMTS13 proteolysis.Previous studies have demonstrated that VWF expressing different blood groups exhibit altered rates of proteolysis by ADAMTS13 (O ≥ B > A ≥ AB). Since α2-6 linked sialic acid and ABO(H) determinants are both expressed as terminal antigens on VWF N-linked glycans, the effect of desialylation upon blood group-specific VWF proteolysis by ADAMTS13 was determined. As expected, untreated group O VWF was cleaved significantly faster than group AB-VWF (p<0.05). However, the ability of ABO blood group to regulate ADAMTS13 proteolysis was completely ablated upon VWF desialylation, as both Neu-O-VWF and Neu-AB-VWF were cleaved by ADAMTS13 at identical rates. This indicates that VWF sialylation constitutes a more important determinant of susceptibility to ADAMTS13 proteolysis than ABO(H) expression.Sialic acid can mediate protein-protein interactions through either conformational and/or charge-mediated mechanisms. Despite this, sodium metaperiodate treatment of pdVWF to remove sialic acid anionic charge did not influence the rate of proteolysis by ADAMTS13. In contrast, the ability of sialic acid to specifically enhance ADAMTS13 proteolysis of VWF was significantly attenuated at high urea concentrations (≥2M), supporting the hypothesis that VWF sialylation enhances proteolysis by ADAMTS13 by promoting a ADAMTS13-specific permissive conformation.These novel data demonstrate that although sialic acid protects VWF against proteolysis by serine and cysteine proteases, it also specifically enhances susceptibility to proteolysis by ADAMTS13. Moreover, the magnitude of this sialic acid-specific effect on VWF proteolysis by ADAMTS13 is more marked than that attributable to N-linked ABO(H) blood group antigen expression. Therefore, quantitative variation in VWF sialylation represents a key regulator of VWF multimeric composition, and as such, is likely to be of clear patho-physiological significance. Disclosures:No relevant conflicts of interest to declare.

Enzymic synthesis of levan and fructo-oligosaccharides by Bacillus circulans and improvement of levansucrase stability by carbohydrate coupling

Bacillus circulans was able to produce extracellular levansucrase using sucrose as carbon source optimally at 35°C. The enzymic synthesis of levan and fructo-oligosaccharides was studied using a 50% ethanol fraction of crude extract. The molecular weight of the synthesized levan was markedly affected by sucrose concentration, the molecular weight of levan decreased with increased sucrose concentration up to 32% whereby fructo-oligosaccharides were isolated. Temperature and the reaction time clearly affected the conversion of fructose to levan with molecular weight values ranging from 10 to 38 kDa. Identification of levan indicated that fructose was the building unit of the levan obtained. Thermal and pH stabilities of B. circulans levansucrase could be improved by enzyme glycosylation using sodium metaperiodate treatment. Chemical modification provides additional points of attachment of the enzyme to the support which offered the modified enzyme greater stabilization than did the free enzyme. The modified enzyme exhibited thermal tolerance up to 50°C, where it retained 88.25% of its activity, while the free enzyme only retained 64.55% of its original activity. The half-life significantly increased from 130 min for the free enzyme to 347 min for the modified enzyme at 50°C, however, it increased from 103 min for the free enzyme to 210 min for the modified enzyme at 60°C. Other properties i.e., the response to some metal ions as well as the ability to convert higher substrate levels and tolerance to an extension of the reaction periods were also improved upon modification. Obviously, the results obtained outlined the conditions leading to the formation of important high or low molecular weight or levan and fructo-oligosaccharides suitable for different industrial applications.

The N-terminal half part of the oral streptococcal antigen I/IIf contains two distinct binding domains.

In order to investigate the binding properties of the antigen I/IIf from Streptococcus mutans, we analyzed the binding activity of five I/IIf derivatives expressed by I/IIf gene derivatives obtained by insertion of a kanamycin resistance marker. ELISA-derived binding assays showed that the derivatives containing both the N-terminal alanine-rich domain (A-region) and an A-region distal domain extending to amino-acid 766 were the most effective in binding biotinylated (Biot-) human salivary components (SAC) and Biot-epithelial cell membrane components. Sodium metaperiodate treatment of SAC inhibited these interactions, suggesting a binding specificity of the A-region distal domain for carbohydrate residues. All the I/IIf derivatives were found to bind Biot-type I collagen, Biot-laminin, Biot-keratin, and Biot-fibronectin, the derivatives containing the A-region but lacking the A-region distal domain exhibiting the highest binding levels. Sodium metaperiodate treatment of laminin had no effect on its binding to the derivatives, suggesting that carbohydrate residues of the ligand were not involved.

Schistosome larvae stimulate macrophage cytokine production through TLR4-dependent and -independent pathways.

Exposure of the mammalian host to infective larvae of Schistosoma mansoni causes an acute inflammatory response in the skin and the activation of several cell types of the innate immune response including macrophages. Using an in vitro model of macrophage activation, we show that schistosome larvae possess molecules that directly stimulate both thioglycollate-elicited macrophages (tM) and IFNgamma-activated tM in vitro to produce several cytokines including IL-6, IL-12p40 and IL-10. The parasite-derived molecules are enriched within the material released by the parasite following transformation [0- to 3-h released larval preparation (0-3hRP)] but not within soluble preparations of whole larvae. Cytokine production was maintained in the presence of polymyxin B, confirming that contaminating endotoxin was not responsible. IL-12p40 and IL-10 production was much lower by cells from C3H/HeJ mice, which have defective Toll-like receptor 4 (TLR4), but IL-6 production was unaffected. Experiments using TLR4-/- mice confirmed that IL-12p40 production by tM in response to 0-3hRP was partly dependent upon functional TLR4, whereas IL-6 production was entirely independent. In contrast, tM from MyD88-/- mice failed to secrete either IL-12p40 or IL-6, underlining a pivotal role of TLR signalling in cytokine production by macrophages in response to stimulation with 0-3hRP. Finally, we show that glycan components of 0-3hRP are required for optimal cytokine production since protease treatment of 0-3hRP had no effect on IL-12p40 production and only a slight effect on IL-6, while sodium meta-periodate treatment almost completely abolished production of both cytokines.

Role of peroxidase inhibition by insulin in the bovine thyroid cell proliferation mechanism.

Monolayer primary cultures of thyroid cells produce, in the presence of insulin, a cytosolic inhibitor of thyroid peroxidase (TPO), lacto peroxidase (LPO), horseradish peroxidase (HRPO) and glutathione peroxidase (GPX). The inhibitor, localized in the cytosol, is thermostable and hydrophylic. Its molecular mass is less than 2 kDa. The inhibitory activity, resistant to proteolytic and nucleolytic enzymes, disappears with sodium metaperiodate treatment, as an oxidant of carbohydrates, supporting its oligosaccharide structure. The presence of inositol, mannose, glucose, the specific inhibition of cyclic AMP-dependent protein kinase and the disappearance of peroxidase inhibition by alkaline phosphatase and alpha-mannosidase in purified samples confirms its chemical structure as inositol phosphoglycan-like. Purification by anionic interchange shows that the peroxidase inhibitor elutes like the two subtypes of inositol phosphoglycans (IPG)P and A, characterized as signal transducers of insulin action. Insulin significantly increases the concentration of the peroxidase inhibitor in a thyroid cell culture at 48 h. The addition of both isolated substances to a primary thyroid culture produces, after 30 min, a significant increase in hydrogen peroxide (H2O2) concentration in the medium, concomitantly with the disappearance of the GPX activity in the same conditions. The presence of insulin or anyone of both products, during 48 h, induces cell proliferation of the thyroid cell culture. In conclusion, insulin stimulates thyroid cell division through the effect of a peroxidase inhibitor, as its second messenger. The inhibition of GPX by its action positively modulates the H2O2 level, which would produce, as was demonstrated by other authors, the signal for cell proliferation.

Identification of a Treponema pallidum laminin-binding protein.

Host extracellular matrix (ECM) components represent ideal microbial adhesion targets that many pathogens use for colonization of tissues and initiation of infection. This study investigated the interaction of the spirochete Treponema pallidum with the ECM component laminin. To identify candidate laminin-binding adhesins, the T. pallidum genome was analyzed to predict open reading frames that encode putative outer membrane proteins, as these proteins interact directly with host ECM components. Subsequent recombinant expression of these proteins and analysis of their laminin-binding potential identified one protein, Tp0751, that demonstrated specific attachment to laminin. Tp0751 attached to laminin in a dose-dependent, saturable manner but did not attach to the ECM component collagen type I or IV or to the negative control proteins fetuin or bovine serum albumin. Sodium metaperiodate treatment of laminin reduced the Tp0751-laminin interaction in a concentration-dependent manner, suggesting that oligosaccharides play a role in this interaction. In addition, Tp0751-specific antibodies were detected in serum samples collected from both experimental and natural syphilis infections, indicating that Tp0751 is expressed in vivo during the course of infection. Collectively, these experiments identified Tp0751 as a laminin-binding protein that is expressed during infection and may be involved in attachment of T. pallidum to host tissues.

A novel tumor-associated mucin of gastrointestinal carcinoma.

To identify a new tumor-associated antigen, a monoclonal antibody, SC142, was produced by immunizing mice with a stomach cancer cell line. The tumor specificity of mAb SC142 was studied by immunohistochemical staining, and the biochemical characteristics of this new gastrointestinal tumor-associated antigen were also studied. The expression of SC142-reactive antigen was investigated in various cancers by immunohistochemical staining. The SC142-reactive antigen was characterized by immunoblotting, sodium metaperiodate treatment assay, O-glycanase digestion assay, and lectin binding assay. The SC142-reactive antigen was highly expressed in 78% of gastric cancers (29/37) and 87% of colon cancers (27/31). No normal colon or stomach tissues remote from the tumor were positive for the antigen. The antibody also reacted with other tumors of epithelial origin such as lung squamous cell cancer (2/4), breast ductal cancer (2/20), bladder transitional cell carcinoma (4/6), and uterine cancer (3/16). Western blot analysis of the antigen revealed glycoprotein(s) which migrated as a smear ranging from the origin of the gel to about the 80 kDa region. The reactivity of this antigen with SC142 was reduced by sodium metaperiodate treatment or O-glycanase digestion, but not by N-glycanase, suggesting that the epitope is an O-glycan. In lectin-binding assay, this antigen reacted only with wheat germ agglutinin but not with Ricinus communis agglutinin, Datura stramonium agglutinin, and Sambucus nigra agglutinin. Our findings indicate that the antigen defined by SC142 is a tumor-associated antigen that could differentiate the gastrointestinal cancer cells from the normal cells. Therefore, SC142 may become a valuable tool for the immunohistochemical diagnosis and tumor immunoscintigraphy of the gastrointestinal cancer patients.

Structure and biological activities of hypochlorite oxidized zymosan

Zymosan (ZYM), a strong complement activating yeast cell preparation, is also a potent inflammatory substance, which shows immunopharmacological activity. Major component of ZYM is β-glucan but contains other constituents, such as mannan, protein, and nucleic acid. We applied sodium hypochlorite (NaClO) treatment to ZYM to reduce impurities and compared the activity with native/parent ZYM. Oxidized ZYM (OX-ZYM) became a nitrogen-free agent. By NMR analysis of native OX-ZYM and zymolyase (endo-1,3-β-glucanase) digest, OX-ZYM was found to contain 1,3-β-linked and 1,6-β-linked glucan moieties, while the latter degraded by sodium metaperiodate treatment. OX-ZYM also contained small amounts of anionic groups, partly reducible by sodium borohydride. Degree of polymerization (DP) of 1,6-β-glucan moiety was estimated to be about DP10–DP50 by MALDI-TOF-MS analysis. In comparison with ZYM activities, OX-ZYM and derivatives showed strong antitumor activity to solid form of Sarcoma 180 in mice, and showed strong activity on alternative pathway of complement, but lost secondary response to ZYM-immune mice. These facts strongly suggested that a particulate form of β-glucan was prepared by NaClO treatment of ZYM and at least a part of ZYM-mediated biological activity was found unmediated by β-glucan moiety.

Schistosoma mansoni: Immunodiagnosis Is Improved by Sodium Metaperiodate Which Reduces Cross-reactivity Due to Glycosylated Epitopes of Soluble Egg Antigen

Alarcón de Noya, B., Colmenares, C., Lanz, H., Caracciolo, M. A., Losada, S., and Noya, O. 2000. Schistosoma mansoni: Immunodiagnosis is improved by sodium metaperiodate which reduces cross-reactivity due to glycosylated epitopes of soluble egg antigen. Experimental ParaIsitology95, 106–112. ELISA with soluble egg antigen (SEA) from Schistosoma mansoni is widely used in the diagnosis of schistosomiasis, but cross-reactivity with other intestinal helminths, overestimating the true prevalence, represents a great limitation. The role of glycoproteins of SEA in cross-reactions was investigated. SEA was oxidized with sodium metaperiodate (SMP) in ELISA and immunoblot. One hundred schistosomiasis-negative individuals sera were submitted to SMP-ELISA improving the specificity from 73% without SMP treatment to 97% with SMP. On the other hand, 94 S. mansoni-positive sera were evaluated showing that 99% were positive in ELISA either with or without SMP treatment, indicating the maintenance of high sensitivity under SMP treatment. By immunoblot, 24 sera from persons with schistosomiasis and 10 sera from schistosomiasis-free persons were assayed under reducing and nonreducing conditions with or without SMP, looking for specific infection markers and cross-reactivity markers. Reactivity from positive sera showed that specific molecules were mainly low-molecular-mass antigens and seem to have predominant proteic epitopes. The unspecific molecules reacting with some schistosomiasis-negative individuals harboring other intestinal parasites (false-positive sera) were mostly larger than 60 kDa and seemed to be basically glycosylated. Glycosylated epitopes have an important role in cross-reaction and SMP can successfully be used to reduce the false reactivity of SEA with no decrease in sensitivity, especially in ELISA as an immunodiagnostic screening surveillance method, which is useful in areas of low schistosomiasis transmission.

Characterization of the adhesion of Flavobacterium columnare (Flexibacter columnaris) to gill tissue

Abstract Flavobacterium columnare (Flexibacter columnaris) is an important cause of gill and skin disease in freshwater fish species, often causing high mortality. In previous studies, virulence of F. columnare was correlated with the ability to adhere to the gill tissue. To gain insight into the factors responsible for adherence, a gill perfusion model was used. The bacterial cells of the high virulence strain AJS 1 were exposed to various treatments, after which they were added to the organ bath of an isolated gill arch and adherence to the gill tissue assessed. Adherence capabilities were significantly reduced following treatment of the bacteria with sodium metaperiodate or incubating them with d‐glucose, N‐acetyl‐d‐glucosamine, d‐galactose and d‐sucrose. Incubation of the bacteria with trypsin and pronase did not significantly inhibit adherence. The binding sites for F. columnare on the gill tissue were also partially characterised. Treatment of the gill with sodium metaperiodate reduced adhesion, but treatment with pronase or trypsin did not cause any significant reduction, indicating that the major component of the receptor is of carbohydrate nature. Adherence ability of the bacteria correlated well with their haemagglutination capacity using chicken and guinea pig erythrocytes. Higher haemagglutination titres were obtained with the highly virulent strain AJS 1 than with strain AJS 4, a strain with low virulence and adherence capacity. Haemagglutination was partially inhibited after incubation of the bacteria with d‐glucose and N‐acetyl‐d‐glucosamine and after treatment of the bacteria at 41_°C for 10_min (minor heat treatment). It was completely abolished following incubation of the bacterial cells with sodium metaperiodate and intensive heat treatment (65_°C, 25_min). Haemagglutination was also in‐sensitive to pronase and trypsin treatment. Transmission electron microscopy (TEM) revealed that the high virulence strain had a thick capsule (120–130_nm) with a regular, dense appearance, whereas the capsule of the low virulence strain was much thinner (80–90_nm) and less dense. TEM also demonstrated the loss of the capsule of the high virulence strain after treatment of the bacterial cells with minor heat and sodium metaperiodate. These results indicate that a lectin‐like carbohydrate‐binding substance incorporated in the capsule is responsible for the attachment of F. columnare to the gill tissue.

Preparation and characterization of an Actinomyces naeslundii aggregation factor that mediates coaggregation with Porphyromonas gingivalis.

Intergeneric coaggregation is responsible for the complexity of the microbiota in human dental plaque and is believed to be important in the initial bacterial colonization of the human oral cavity. Actinomyces naeslundii, an early colonizer of the tooth surface, may enhance subsequent colonization by Porphyromonas gingivalis which is associated with adult periodontitis. The purpose of this study was to isolate and characterize the A. naeslundii aggregation factor (AnAF) that mediates coaggregation with P. gingivalis. AnAF was isolated from A. naeslundii sonic extract (SE) by gel filtration on a Sephacryl S-400HR, by hydrophobic interaction chromatography on a HiTrap Octyl Sepharose 4FF, and by ion exchange chromatography on a HiTrap Q. The specific activity increased 12-fold with a yield of 2.5%. SDS-PAGE analysis of AnAF revealed a protein band of high molecular weight in excess of 200 kDa. Carbohydrate was detected as the only material coinciding with the protein band, indicating that the AnAF was a glycoprotein. Immunoblotting analysis indicated that AnAF directly bound to P. gingivalis cells. AnAF was sensitive to sodium metaperiodate treatment but not to heat or protease treatments. These results suggest that the AnAF carbohydrate component mediated coaggregation with P. gingivalis cells. AnAF also inhibited coaggregation with other periodontal disease-associated bacteria such as Prevotella intermedia, Fusobacterium nucleatum, Capnocytophaga ochracea, but not streptococci.