Agglutinating Factor Research Articles

Molecular characterization and virulence factor profiles of Staphylococcus aureus isolated from bovine mastitis in Brazilian herds

Staphylococcus aureus is the main etiological agent of bovine mastitis worldwide and knowledge about its diversity and virulence factors is vital in controlling infections caused by this pathogen. The present study aimed to perform molecular characterization of a population of S. aureus (n=153 strains isolated from 1994 to 2014 in seven Brazilian states) by pulsed-field gel electrophoresis (PFGE) and evaluate their virulence profiles via polymerase chain reaction (PCR). PFGE identified 93 pulsotypes, with the isolates organized into 26 clusters and 20 unique pulsotypes. Predominant pulsotypes were observed, with variations according to the years of isolation and geographic origin of the isolates. Based on the PCR results for the genes encoding agglutination factors (ClfA and ClfB), binding proteins (fibronectin binding protein - FnBPA, elastin binding protein - Ebps, collagen binding protein - Cna), and toxins (Hla, Hlb and Luk-ED), 40 virulence profiles were detected. The frequency of virulence genes ranged from 58 to 98% (clfA:84.3%; clfB and hlb both 81%; hla:71.2%; fnBA:82.3%; Can:94.7%; ebps:58%; and lukED:98%). The existence of prevalent genotypes in some of the Brazilian states and the time period studied suggests that these genotypes are better adapted, with favorable characteristics in host/pathogen relationships. Genes of proven importance for S. aureus pathogenesis in bovine mastitis were widely distributed in genetically divergent populations, suggesting that most of these genes may be interesting candidates in the development of vaccines to control bovine mastitis in Brazil.

Evaluation of biocompatible amino acid-functionalized cellulose composites: Characterizations, molecular modeling, anticoagulant activity, and cytocompatibility

This study explored the anticoagulant activity of biocompatibility and biodegradability of all-natural composite based on cellulose cysteine Schiff's base composites and compared it with molecular modeling calculations. These composites were prepared through two steps: cellulose was oxidized to dialdehyde cellulose (DAC) via sodium periodate, followed by coupling with a different ratio of cysteine. The oxidation and coupling reactions were confirmed by studying the chemical structures via physiochemical analysis and surface morphology via scanning electron microscope (SEM) techniques. In addition, the prepared samples' anticoagulant activity was studied by determining blood agglutination factors regarding prothrombin time (PT) and partial thromboplastin time (PTT) compared with the activity of native cellulose and DAC. The obtained results demonstrated that cellulose cysteine Schiff's base composites enhance the coagulation times of blood plasma, and maximum anticoagulant activities were recorded at 45, 40, and 35 μg/mL for loading of 0.4, 0.8, and 1.2 mmoL cysteine, respectively. The cytotoxicity of the prepared composites was also tested using a Vero cell (normal fibroblast cell line). Results revealed that cellulose cysteine Schiff's base composites had negligible cytotoxicity.Additionally, the activity of the prepared composites was performed and referred to as capitulation calcium ion. Moreover, the molecular modeling of bovine thrombin complexes (PDB: 1ETT) with cellulose cysteine Schiff's base was studied to confirm the functionalization of composite toward the calcium ion to prevent blood coagulation. This study suggests using cellulose cysteine Schiff's base composites as a coating in blood packs in the blood donation process.

Generating shared value: intersection between organisations that invest in social innovation

PurposeThis study aims to investigate how organizations that invest in social innovation – such as for-profit and social enterprises – organize and shape the dynamics between social innovation and transformation.Design/methodology/approachThis is a qualitative interpretative study through the theoretical lens of social innovation. The organizational phenomenon studied was the business strategy for sustainable regional development of a Brazilian financial institution. Secondary data were collected from financial institution’s documents. The data analysis followed a categorization technique.FindingsThis research presents five intersectional elements that act as agglutinating factors between the structural elements common to these organizations: the processes (inter-organizational and intra-organizational), the forms of materialization of social innovation (products, experiences and replicable forms) and the impacts (in society and organization).Practical implicationsThe paper contributes to the debate about social innovation as to its definition, showing that it is both a phenomenon and a theory, which is still little explored. In particular, in emerging countries, such as Brazil, which publishes little about social innovation in international literature, going beyond European countries. Second, an empirical deeper understanding of the for-profit “organizations” modus operandi adopts and integrates social innovation in their strategies and operations. Third, given the multidimensional and complex nature of the social systems involved in innovation actions, it discusses a strategy that acts as an agglutinating factor. Finally, with reflections so that managers can discuss and start up a social innovation project.Originality/valueMost research is based on nonempirical evidence. Among the few empirical works, the focus is on the performance of nonprofit organizations in the promotion, development and implementation of projects involving social technology. The literature does not adequately understand how for-profit organizations adopt and integrate social innovation into their strategies and operations. Furthermore, it is observed that research analyzes data from European countries, with few approaches eying countries in development.

Search for host defense markers uncovers an apple agglutination factor corresponding with fire blight resistance.

Pathenogenesis-related (PR) proteins are extensively used as molecular markers to dissect the signaling cascades leading to plant defense responses. However, studies focusing on the biochemical or biological properties of these proteins remain rare. Here, we identify and characterize a class of apple (Malus domestica) PR proteins, named M. domestica AGGLUTININS (MdAGGs), belonging to the amaranthin-like lectin family. By combining molecular and biochemical approaches, we show that abundant production of MdAGGs in leaf tissues corresponds with enhanced resistance to the bacterium Erwinia amylovora, the causal agent of the disease fire blight. We also show that E. amylovora represses the expression of MdAGG genes by injecting the type 3 effector DspA/E into host cells and by secreting bacterial exopolysaccharides. Using a purified recombinant MdAGG, we show that the protein agglutinates E. amylovora cells in vitro and binds bacterial lipopolysaccharides at low pH, conditions reminiscent of the intercellular pH occurring in planta upon E. amylovora infection. We finally provide evidence that negatively charged polysaccharides, such as the free exopolysaccharide amylovoran progressively released by the bacteria, act as decoys relying on charge-charge interaction with the MdAGG to inhibit agglutination. Overall, our results suggest that the production of this particular class of PR proteins may contribute to apple innate immunity mechanisms active against E. amylovora.

RNA-seq: The early response of the snail Physella acuta to the digenetic trematode Echinostoma paraensei.

To analyze the response of the snail Physella acuta to Echinostoma paraensei, a compatible digenetic trematode, Illumina RNA-seq data were collected from snails with early infection (5 snails at 2 days post-exposure [DPE]) and established infection (4 snails, 8 DPE), and 7 control (unexposed) snails. A reference transcriptome (325,563 transcripts, including 98% of eukaryotic universal single-copy orthologs; BUSCO) and a draft P. acuta genome (employing available genomic Illumina reads; 799,945 scaffolds, includes 88% BUSCO genes) were assembled to guide RNA-seq analyses. Parasite exposure of P. acuta led to 10,195 differentially expressed (DE) genes at 2 DPE and 8,876 DE genes at 8 DPE with only 18% of up-regulated and 22% of down-regulated sequences shared between these time points. Gene ontology (GO) analysis yielded functional annotation of only 1.2% of DE genes but did not indicate major changes in biological activities of P. acuta between 2 and 8 DPE. Increased insights were achieved by analysis of expression profiles of 460 immune-relevant DE transcripts, identified by BLAST and InterProScan. Physella acuta has expanded gene families that encode immune-relevant domains, including CD109/TEP, GTPase IMAP, Limulus agglutination factor (dermatopontin), FReD (≥82 sequences with fibrinogen-related domains), and transcripts that combine C-type lectin (C-LECT) and C1q domains, novel among metazoa. Notably, P. acuta expressed sequences from these immune gene families at all time points, but the assemblages of unique transcripts from particular immune gene families differed between 2 and 8 DPE. The shift in profiles of DE immune genes, from early exposure to parasite establishment, suggests that compatible P. acuta initially respond to infection but switch to express immune genes that likely are less effective against E. paraensei but counter other types of (opportunistic) pathogens and parasites. We propose that the latter expression profile is part of an extended phenotype of E. paraensei, imposed upon P. acuta through parasite manipulation of the host, following successful parasite establishment in the snail after 2 DPE.

Evaluation of Sperm Impairing Factor from Serratia marcescens as Male Contraceptive in Mouse Model.

The present study was carried out to assess the contraceptive efficacy of sperm agglutinating factor (SAF) isolated from Serratia marcescens, in male Balb/c mice. Mice were administered via an intratesticular route with different concentrations of SAF, viz., 10, 50, 100, 200, or 400 µg, in the right testis only which served as a test while the left side served as control except otherwise stated. Mice were sacrificed on day 3, 7, 14, 21, 30, 45, 60, and 90 after administration, and results in terms of change in body weight, seminal parameters, tissue somatic indices (TSI), hematological parameters, serum level of testosterone, lipid peroxidation, and histology were studied. The body weight and TSI remained unaffected in all the experimental groups. In case of seminal parameters, the right testis treated with 10 μg, 50 μg, 100 μg, 200 μg, or 400 μg of SAF showed azoospermia up to day 7, 14, 21, 45, and 90, respectively. The hematological indices, aspartate aminotransferase (AST), and alanine aminotransferase (ALT) were found to be unaltered when the group receiving SAF (test) was compared with the groups receiving phosphate buffer saline (control) in the right testis; however, the treatment had a negative effect on the serum level of testosterone. It also affected the oxidative status of the right testis. Furthermore, histological studies revealed hypospermatogenesis and alterations in the seminiferous tubules which included intraepithelial vacuolation and exfoliation in the right side as compared to the left side. Thus, the results suggest that SAF (400 µg) causes suppression of spermatogenesis, without causing apparent toxic effects.

Contraceptive efficacy of sperm agglutinating factor from Staphylococcus warneri, isolated from the cervix of a woman with inexplicable infertility

BackgroundVoluntary control of fertility is of paramount importance to the modern society. But since the contraceptive methods available for women have their limitations such as urinary tract infections, allergies, cervical erosion and discomfort, a desperate need exists to develop safe methods. Vaginal contraceptives may be the answer to this problem, as these are the oldest ways of fertility regulation, practiced over the centuries. With minimal systemic involvement, these are also the safest. Natural substances blocking or impairing the sperm motility offer as valuable non-cytotoxic vaginal contraceptives. Antimicrobial peptides (AMPs) isolated from plants, animals and microorganisms are known to possess sperm immobilizing and spermicidal properties. Following this, in the quest for alternative means, we have cloned, over expressed and purified the recombinant sperm agglutinating factor (SAF) from Staphylococcus warneri, isolated from the cervix of a woman with unexplained infertility.MethodsGenomic library of Staphylococcus warneri was generated in Escherichia coli using pSMART vector and screened for sperm agglutinating factor (SAF). The insert in sperm agglutinating transformant was sequenced and was found to express ribonucleotide-diphosphate reductase-α sub unit. The ORF was sub-cloned in pET28a vector, expressed and purified. The effect of rSAF on motility, viability, morphology, Mg++-dependent ATPase activity and acrosome status of human sperms was analyzed in vitro and contraceptive efficacy was evaluated in vivo in female BALB/c mice.ResultsThe 80 kDa rSAF showed complete sperm agglutination, inhibited its Mg2+-ATPase activity, caused premature sperm acrosomal loss in vitro and mimicked the pattern in vivo showing 100% contraception in BALB/c mice resulting in prevention of pregnancy. The FITC labeled SAF was found to bind the entire surface of spermatozoa. Vaginal application and oral administration of rSAF to mice for 14 successive days did not demonstrate any significant change in vaginal cell morphology, organ weight and tissue histology of reproductive and non-reproductive organs and had no negative impact in the dermal and penile irritation tests.ConclusionThe Sperm Agglutinating Factor from Staphylococcus warneri, natural microflora of human cervix, showed extensive potential to be employed as a safe vaginal contraceptive.

Platelet Surface PDI Controls the Ligand-Binding Function of Glycoprotein Ibalpha and Platelet-Neutrophil Interactions Under Thromboinflammatory Conditions

We previously showed that the isomerase activity of platelet surface protein disulfide isomerase (PDI), a prototypic thiol isomerase, regulates the full activation of αIIbβ3 integrin and is important for platelet accumulation, but not hemostasis, following vascular injury (Kim et al. Blood 2013). However, it remains unclear whether platelet PDI regulates the function of other platelet surface receptors. Since it was reported that platelet PDI and glycoprotein Ibα (GPIbα) were physically close on the platelet surface (Burgess et al. JBC 2000), we investigated whether platelet PDI regulates GPIbα function using megakaryocyte-specific PDI conditional knockout (CKO) mice developed by us. We found that PDI-null platelets showed a significant defect in GPIbα-mediated agglutination and von Willebrand factor (vWF) binding. Those defects were completely rescued by exogenously-added recombinant wild-type PDI (wtPDI), but not mutant PDI (dmPDI) lacking the isomerase activity. Consistently, inhibition of platelet PDI with blocking antibodies impaired GPIbα-mediated agglutination and vWF binding in human and mouse platelets, suggesting that the isomerase activity of platelet surface PDI controls the ligand-binding function of GPIbα. Studies using surface plasmon resonance revealed that wtPDI and dmPDI bound to immobilized GPIbα with a dissociation constant of 0.9 and 1.2 µM, respectively. Mass spectrometric analysis indicated that the Cys4-Cys17 disulfide bond in GPIbα, which has an allosteric -RHStaple configuration, was preferentially reduced by wtPDI, but not dmPDI. Consistent with previous studies showing that platelet GPIbα is important for platelet-neutrophil interactions, we observed that deletion of platelet PDI significantly reduced platelet-neutrophil interactions under shear conditions. Importantly, using real-time fluorescence intravital microscopy with megakaryocyte-specific PDI CKO mice, we demonstrated that platelet-specific PDI deletion did not influence neutrophil adhesion but markedly reduced platelet adhesion and accumulation on the adherent neutrophils on TNF-α-inflamed cremaster muscle venules. Infusion of wtPDI, but not dmPDI, restored reduced platelet-neutrophil interactions in the PDI CKO mice, implying the importance of platelet surface PDI activity in regulating GPIbα-mediated platelet-neutrophil interactions. Using a thromboinflammation model of hepatic ischemia/reperfusion injury, we found that platelet-specific PDI CKO mice exhibited a significant reduction in the infarct size and the serum levels of aspartate aminotransferase, a marker of liver damage. Our results suggest that the isomerase activity of platelet surface PDI regulates the redox state of the Cys4-Cys17 disulfide bond in GPIbα and is required for the ligand-binding function of GPIbα, thereby playing a critical role during platelet-neutrophil interactions under thromboinflammatory conditions. DisclosuresNo relevant conflicts of interest to declare.

N-Acetylglucosaminylation of Serine-Aspartate Repeat Proteins Promotes Staphylococcus aureus Bloodstream Infection

Staphylococcus aureus secretes products that convert host fibrinogen to fibrin and promote its agglutination with fibrin fibrils, thereby shielding bacteria from immune defenses. The agglutination reaction involves ClfA (clumping factor A), a surface protein with serine-aspartate (SD) repeats that captures fibrin fibrils and fibrinogen. Pathogenic staphylococci express several different SD proteins that are modified by two glycosyltransferases, SdgA and SdgB. Here, we characterized three genes of S. aureus, aggA, aggB (sdgA), and aggC (sdgB), and show that aggA and aggC contribute to staphylococcal agglutination with fibrin fibrils in human plasma. We demonstrate that aggB (sdgA) and aggC (sdgB) are involved in GlcNAc modification of the ClfA SD repeats. However, only sdgB is essential for GlcNAc modification, and an sdgB mutant is defective in the pathogenesis of sepsis in mice. Thus, GlcNAc modification of proteins promotes S. aureus replication in the bloodstream of mammalian hosts.

Receptor mediated amelioration of the detrimental effects of sperm agglutinating factor on sperm parameters

Sperm agglutinating factor (SAF) isolated from Staphylococcus aureus immobilizes spermatozoa by agglutination and causes sperm death. This interaction of SAF with spermatozoa is receptor mediated and this receptor has been isolated and purified from human spermatozoa. In this study we attempt to study whether the receptor could ameliorate the detrimental effects of SAF on sperm parameters. Receptor was evaluated against SAF mediated compromised sperm parameters such as Mg(2+) dependent ATPase activity, acrosome status and apoptosis, in vitro using fluorescent microscopy and flow cytometry as well as in vivo by studying the impact on fertility in mice. Incubation of SAF (80 μg) with spermatozoa resulted in reduced Mg(2+) dependent ATPase activity and premature acrosomal loss whereas a higher concentration (100 μg), induced apoptosis. However, in the presence of receptor a dose dependent blockage of SAF induced inhibition of Mg(2+) dependent ATPase activity was observed. At higher concentrations 100 and 125 μg, receptor could inhibit both the premature acrosomal loss and apoptosis. In vivo studies showed that receptor (50 μg) could alleviate SAF induced infertility in female Balb/c mice following a single intravaginal application before mating. The work highlights the efficacy of the receptor as a corrective measure against negative influence of SAF on functional parameters of spermatozoa as well as fertility and presents receptor as a potential therapeutic intervention against SAF induced infertility.

Characterization of sperm agglutinating factor isolated from staphylococcus aureus and its corresponding receptor from spermatozoa

Spermagglutination factor isolated from Staphylococcus aureus showed spermagglutinating and spermicidal properties in vitro. In an attempt to characterize SAF, it was tested for the various factors produced by S. aureus viz. catalase, coagulase, hemolysins, protease, lipase, phospholipase C, nuclease and protein A. As, SAF did not show any of these activities, therefore to identify it, peptide mass fingerprinting using Matrix Assisted Laser Desorption Ionization-Time of flight (MALDI-TOF) was done. Matching of the mass spectrum in NCBI database showed sequence homology with hypothetical protein BACPEC_00178 of Bacteroides pectinophilus ATCC 43243. SAF conjugated with FITC was able to bind to spermatozoa indicating the presence of receptor on sperm surface. Calorimetric analysis of SAF and this purified receptor interaction showed the binding constant, enthalpy of binding ΔH° as 1130/M and -11.6kJ/mole while the values of free energy and entropy were -18.1kJ/mole and 20.9J/moleK, respectively. The receptor showed sequence similarities with chain A crystal structure of Ga module complexed with human serum albumin when subjected to MALDI-TOF.

Amelioration of Sperm Agglutinating Factor (SAF) Induced Sperm Impairment by Anti-SAF Polyclonal Antibody

Background: We have previously isolated a Spermagglutinating Factor (SAF) from Escherichia coli, which was capable of causing sperm agglutination and impairment of sperm parameters viz. apoptosis, premature acrosome loss and inhibition of Mg2+ dependent ATPase activity invitro. In addition, intravaginal administration of Balb/c mice with SAF resulted in infertility. To provide evidence that SAF plays an important role in impairment of sperm parameters and infertility, anti-SAF antiserum was raised and its application as a therapeutic intervention against SAF induced damage was evaluated. Methods: Effect of anti-SAF antiserum was evaluated against SAF mediated adverse effects on sperm parameters. Sperm agglutination was observed using light microscopy and Mg2+ dependent ATPase activity was estimated in terms of release of inorganic phosphate. Sperm apoptosis and acrosome status were evaluated by means of flow cytometery and fluorescent microscopy, respectively. Further, the impact of anti-SAF antiserum was also seen on fertility outcome in mice Results: Results showed that immunization of mice with SAF lead to the generation of high titer specific antibodies. Raised anti-SAF antiserum could neutralize all the biological effects of SAF in contrast to control antiserum. Furthermore, intravaginal application of anti-SAF antiserum along with SAF rendered mice fertile. Conclusion: Here we provide evidence that SAF plays an imperative role as all the detrimental effects induced by SAF whether in vitro or in vivo were blocked on simultaneous incubation with anti-SAF antiserum. Present work also highlighted the efficacy of the anti-SAF antiserum as a curative measure against SAF.

Epidemiology, Diagnosis, and Management ofvon Willebrand Disease in India

Von Willebrand disease (VWD) in all developing countries including India is considered a rare coagulation disorder, contrary to many reports from Western countries. Prevalence data based on hospital referrals identifies type 3 VWD as the most common subtype followed by type 1 and type 2. Approximately 60 to 70% cases of type 3 VWD are reportedly born of consanguineous marriages. The discriminatory diagnostic tests mainly include assays for factor (F)VIII:C and ristocetin-induced platelet agglutination and Von Willebrand factor (VWF) antigen either by immunoelectrophoresis or by enzyme-linked immunosorbent assay. VWD-type assisting tests like VWF collagen binding, VWF ristocetin cofactor assay, VWF-FVIII binding assay, and multimer analysis are occasionally used but not routinely applied in many laboratories. Among women, menorrhagia is an important presenting manifestation. Except for a handful of centers mainly in metropolitan cities, most laboratories in the remote parts of the country have no facilities for VWD-related investigations, resulting in occasional misdiagnoses of VWD as hemophilia A. Genetic diagnosis is being offered in two or three centers using the indirect linkage method in type 3 VWD, and efforts are continuing to implementing a direct mutation detection technique for routine practice in a few laboratories. Depending on the subtype or the severity of VWD, desmopressin, cryoprecipitate, fresh-frozen plasma, and factor VIII/VWF concentrates are used for management. Antifibrinolytic agents like epsilon-aminocaproic acid and tranexamic acid are widely used as an adjuvant therapy. In women with menorrhagia, oral contraceptives as a supplementary treatment are also being widely advocated to reduce bleeding. Products like danazol, ethenyl estradiol, thalidomide, and atorvastatin have been used in individual patients; acquired VWD associated with hypothyroidism has been managed successfully with thyroid hormone treatment. Both minor and major surgical procedures are performed in a few centers with judicious use of cryoprecipitate or FVIII concentrate containing VWF along with other supplementary therapeutic products to achieve adequate hemostasis. Awareness about the disease, establishment of the comprehensive coagulation laboratory, and treatment centers will be successful in increasing diagnosis of VWD and consequently better management of affected patients. This is likely to tilt the ratios of different VWD types, and VWD is likely to emerge as the most common of all coagulation disorders in the near future.

Immunization strategies and enhanced immunity of typhoid vaccine using potato starch polymer as an adjuvant

Controlled release of typhoid antigen encapsulated potato starch microparticles minimize the disadvantages in terms of cost, multiple doses and side effects. The development of this work may enable to replace the alum to elicit sustained immune response because alum induces local granuloma and hypersensitivity reaction to some individuals. We have employed emulsion crosslinking technique by using 0.5% ml glutaraldehyde as a crosslinking agent. The particle size was analyzed as 43.34 µm; the loading capacity was about 44.76% and the loading efficiency as about 75.17%, while the morphology was unchanged in the stability studies. In vitro studies were analyzed by SEM and compatibility studies were performed by IR α F.T spectroscopic analysis. The percentage of antigenically active typhoid antigens was found to be 91.5% by ELISA after entrapment with 8% of starch and 0.5% of stabilizing agent. Immunoprecipitation assay exhibited that the amount of antigen in the sample was found to be 51µg and at the dilution of 1:50 showed agglutination factor (with the titer value of 50). Keywords: Typhoid, potato starch, biodegradability, immunomodulatory, single therapy, antibody response.

Safety studies of sperm agglutinating factor produced by Staphylococcus aureus as a vaginal contraceptive: in vivo studies

Sperm agglutinating factor (SAF) isolated from Staphylococcus aureus when applied at concentration 10 μg before mating completely prevented conception in the mouse. The objective of the present study was to evaluate its safety, as safety is an important concern to be addressed before a compound is selected for contraceptive use. Our results showed that SAF has a very high safety profile. Vaginal application of SAF at 10 μg to the mouse for 14 consecutive days caused no systemic toxicity and vaginal irritation as indicated by lack of effect on organ weights and histology. Moreover, no adverse effect was observed on the subsequent reproductive capability, peritnatal outcome and growth and development of the offspring. SAF (10 μg) did not irritate the skin or penile mucosa. Oral administration of 2 mg/kg body weight of SAF did not show any toxicity to reproductive and non-reproductive organs. Therefore, SAF with spermicidal activity and lack of toxicity may have the potential to become the active ingredient of a vaginal contraceptive.

Immunization Strategies and Enhanced Immunity of Typhoid Vaccine Using Potato Starch Polymer as an Adjuvant

Controlled release of typhoid antigen encapsulated potato starch microparticles minimize the disadvantages in terms of cost, multiple doses and side effects. The development of this work may enable to replace the alum to elicit sustained immune response because alum induces local granuloma and hypersensitivity reaction to some individuals. We have employed emulsion crosslinking technique by using 0.5% ml glutaraldehyde as a crosslinking agent. The particle size was analyzed as 43.34 µm; the loading capacity was about 44.76% and the loading efficiency as about 75.17%, while the morphology was unchanged in the stability studies. In vitro studies were analyzed by SEM and compatibility studies were performed by IR α F.T spectroscopic analysis. The percentage of antigenically active typhoid antigens was found to be 91.5% by ELISA after entrapment with 8% of starch and 0.5% of stabilizing agent. Immunoprecipitation assay exhibited that the amount of antigen in the sample was found to be 51μg and at the dilution of 1:50 showed agglutination factor (with the titer value of 50).

Receptor Mediated Agglutination of Human Spermatozoa by Spermagglutinating Factor Isolated From Staphylococcus aureus

We examined spermagglutinating factor isolated from Staphylococcus aureus for evidence of receptor mediated agglutination of human spermatozoa. Binding to spermatozoa by spermagglutinating factor isolated from S. aureus with a high degree of specificity indicates receptor-ligand interaction. To examine this interaction we isolated and purified the ligand and the receptor. To assess receptor mediated agglutination of spermatozoa further we blocked spermagglutination induced by spermagglutinating factor in the presence of receptor. Spermagglutinating factor induced spermagglutination was competitively inhibited by adding purified receptor, indicating that sperm agglutinating factor isolated from S. aureus attaches to specific receptors on human spermatozoa. The spermagglutinating factor receptor was a protein with a molecular weight of approximately 57 kDa. Spermagglutinating factor induced spermagglutination and at higher concentrations had a spermicidal effect, which was inhibited by introducing the receptor. As observed on scanning electron microscopy studies, incubating spermatozoa with spermagglutinating factor showed profound morphological alterations. However, spermatozoa with normal morphology were noted when incubated with spermagglutinating factor in the presence of receptor, indicating that morphological alterations may account for spermatozoa agglutination by spermagglutinating factor. Results suggest that spermagglutinating factor isolated from S. aureus may bind specifically to sperm surface receptor sites before causing spermagglutination.

Aspirin Sensitive Asthma: Provocation Tests Do Not Work in Such Patients

In response to the impressive scientific review article by Randerath and Galetke, we want to share our own experiences with the treatment of asthma and nasal polyps, in these times of increasing funding shortages, to show that there are simpler solutions to the problem. Small practices cannot present high case numbers or evidence- based study designs. We tested the tolerability of salix (willow) and acetylsalicylic acid (ASA) in 536 patients with allergies using electroacupuncture according to the method of Voll (EAV) in 115 patients with rhinoconjunc tivitis and in 65 asthma patients with concomitant migraine or lipid metabolism disorders (1). Of those 180 patients 11 were affected, of whom two reacted to willow and ASA. They were successfully desensitized by using destructive interference (3). This method is used similarly for stimulants such as tree or grass pollen, house dust mites, eiderdown, and synthetic fibers, to prevent nasal polyps and subsequent aspirin sensitive asthma successfully. The author has been using ASA at low dosages since 1985 after testing for thrombocyte hyperaggregability (2). Platelet agglutinating factor (PAF) is released in every allergic reaction and causes microcirculatory disruptions that may manifest as pain or shortness of breath. Mast cells are activated not only by IgE but also by type III reactions (IgG). Immunocomplexes develop that activate complement pathways, thereby releasing slow reacting substances or leukotrienes. The simultaneously released heparin and similar glycosaminoglycans stimulate the matrix to release large basic proteins and arginine and lysine from eosinophils, which pro vides a stimulus to attract more eosinophils. These processes are disrupted by destructive interference, and the mast cell system can calm down. We cannot accept Randerath and Galetke’s statement: Currently there is no reliable in-vitro method to diag nose aspirin sensitive asthma, so that provocation tests are vital. Our own clinical practice has shown that prov ocation tests do not work in such patients. We there fore need leukotriene antagonists for such patients only in the initial phase of their treatment. ASA does not have to present a real problem in practice.

Histomonas meleagridis (Parabasala, Trichomonadea, Monocercomonadidae): presence of natural agglutinins in horse serum

Cultured Histomonas meleagridis cells were readily agglutinated in vitro by horse serum at concentrations as low as 5%, although clumping was more rapid and prominent at 15% or higher. For observation of clumping, the cultured organisms were washed twice in Hanks balanced solution (HBSS) by centrifugation (1,000 x g for 15 min) and filtered through glass wool. The test sera were added and the mixture incubated in a Petri plate or 24-well culture plates at r.t. for 15-30 min. Formation of clumps was time- and concentration-dependent. Gentle agitation hindered agglutination at low serum concentration and accelerated agglutination at higher concentrations. The agglutinating factor (AF) was detected in several batches of serum from different sources, regardless of whether sera were heat-treated to inactivate complement. Histomonads were not clumped by either fetal horse or bovine serum (5-30%). Neither chicken nor turkey serum agglutinated histomonads to the extent seen with horse serum. Immune turkey serum lysed histomonads, hindering observation of clumping. Complement inactivation of immune serum slightly reduced lysis. AF in horse serum was precipitated with 25-40% ammonium sulfate and was active when cleaned by dialysis and reconstituted in HBSS. Clumping by serum facilitated the cleaning of histomonads for other studies where pure suspensions were needed.

Biochemical Characterization of C-type Lectin in the Diamondback Moth, Plutella xylostella (Yponomeutidae: Lepidoptera)

Lectins due to their affinity to carbohydrate moiety are involved in diverse functions like cell attachment in embryogenesis, organogenesis and cellular trafficking as well as nonself recognition in immune responses. Agglutinating activity was detectable in Plutella xylostella (Yponomeutidae: Lepidoptera) against 14 different species including bacterial and yeast cells, among which the whole body homogenate of P. xylostella agglutinated Providencia vermicola, Flavobacterium sp., and Saccharomyces cerevisiae with high titers. On analysis of physico-chemical properties, this putative agglutinating factor (s) was specifically dependent on the presence of Ca ++ for its activity and was reversibly sensitive to EDTA. The agglutinating activity was stable at pH 6–8, but was heat-labile. The agglutinating factor (s) was proteinaceous in nature as it was completely precipitable by ammonium sulphate. Its carbohydrate binding activity was demonstrated by inhibition assay, which revealed that methyl α-D-mannopyranoside inhibited agglutination against P. vermicola. In contrast, P. xylostella parasitized by an endoparasitoid wasp, Cotesia plutellae (Braconidae: Hymenoptera), also showed the agglutination properties with somewhat higher activity than the nonparasitized. Carbohydrate inhibition assay with methyl α-D-mannopyranoside was detectable at one-fold higher concentration in the homogenate of the parasitized larvae, suggesting that the agglutinating factor (s) is inducible or due to de novo parasitism-specific synthesis. These results suggest the presence of calcium-dependent lectin in P. xylostella and an alteration in the agglutinating property by C. plutellae parasitization.