The role of non-specific serum opsonins in thein vitro interaction (adherence) between peritoneal macrophages of newborn precolostral piglets and rough strain ofEscherichia coli

The role of non-antibody, natural opsonins in sera of newborn, precolostral piglets for the early phase of phagocytosis (adhesion) of roughEscherichia coli to peritoneal macrophages of these animals, was studied. Suspensions of macrophages, incubated together with bacteria in the presence or absence of piglet serum opsonins, were submitted to differential centrifugation to enable the separation of macrophage-associated bacteria (i.e. opsonized) from unopsonized, free bacteria in the supernatant. It was found that native piglet sera having no detectable antibody activity toEscherichia coli, do possess significant opsonic activity,i.e. they enhanced thein vitro adherence of roughEscherichia coli to macrophages. Furthermore, this activity could be removed by procedures or substances known to inactivate the complement by different mechanisms: heating for 30 min at 56°C, addition of EDTA, absorption of sera by zymosan, addition of complement inhibitors phosphomannan and carrageenin. These results are interpreted as further evidence for the presence of complement-dependent serum opsonins to roughEscherichia coli in sera of newborn precolostral piglets.     

Comparative characteristics of conglutination and immunoconglutination reactions and their participating constituents]

The difference between the hemolytic and conglutinating properties of equine complement was shown by the method of conglutinin and immunoconglutinin titration in the author's modification. Different localization of conglutinating antibodies (in the supernatant fraction) and conglutinins (in the sediment) in bovine serum was revealed by the method of serum fractionation in the stream of carbon dioxide. The differences in the localization of conglutinins and immunoglutinins (in the sediment and the supernantant fraction respectively), as well as the differences in the localization of hemagglutinins and conglutinins in the sera (in different fractions) were determined.     

Prokaryotic Expression of Ovis aries Conglutinin Encoding Neck and Carbohydrate Recognition Domain and its Functional Characterization

Conglutinin, a soluble pattern recognition receptor of innate immune system in bovines is known for its potential defensive activity against microorganisms either by direct agglutination in the presence of calcium or by acting as opsonin. In the present study, sheep (Ovis aries) conglutinin encoding neck and carbohydrate recognition domain (rSCGN) was expressed in the E coli BL21 expression host. The recombinant conglutinin revealed molecular weight of 27 kDa in SDS PAGE and also in western blotting using antibuffalo conglutinin polyclonal serum. The protein was characterized further for its functional activity in various assays. In ELISA based sugar and LPS binding assay, the rSCGN revealed its high binding activity toward N-acetyl glucosamine and E. coli LPS in the presence and the absence of calcium ions, respectively. Hemagglutination of chicken red blood cells caused by Newcastle disease virus was not inhibited in the presence of rSCGN as it lacked complete collagenous region present in the native protein. In virus neutralization test, the recombinant protein was found to reduce multiplication of bovine herpes virus-1 propagated in MDBK cells. This prokaryotically expressed 27 kDa recombinant sheep conglutinin can serve as antigen in future studies to develop sandwich ELISA for assessing the level of native conglutinin in sheep serum.     

The relation between the bactericidal activity of complement and the character of the bacterial surfaces

The bactericidal activity of sera not containing antibodies (sera from precolostral piglets and calves) was tested with strains of gram-negative bacteria with different surfaces. The accuracy of the method of bactericidal test was evaluated statistically the bactericidal unit of complement was defined for comparing the activity of sera of different animals and different species. Various methods used for estimating the character of bacterial surface were compared. It was found that the bactericidal activity of piglet sera is directly dependent on the content of complement in the sera tested and the character of the bacterial surface (in the R-form). In selected strains there is a correlation in all criteria characterizing the surfaces of bacteria, and their susceptibility to bactericidal activity of sera; in a group of 37 strains selected at random, correlation with only one of the surface characteristics (stability in solution after heating to 100°C for 1 hour) was found. In calf sera a component was found which increases the effect of complement to some strains (e.g.Shigella shigae). This component may by absorbed from the serum only in the presence of complement. The nature of this factor is discussed.     

Neutralization of bacterial viruses by antibodies of young animals

The cofactor activity of complement of precolostral newborn pig serum was compared with the haemolytic and bactericidal activity. Using current decomplementation treatment it was found that all three types of activity are destroyed in parallel i.e. haemolytic, bactericidal and cofactor (heat inactivation, EDTA, ammonia, trypsin, adsorption to zymosan or immune precipitate). In an ultracentrifugation analysis the haemolytic, bactericidal and cofactor activities showed the same sedimentation pattern. Using gel filtration on a Sephadex G 200 column, the cofactor activity was found in the fraction which contained all basic complement components. On the basis of these results it can be concluded that the complement of piglet serum acts as a factor which potentiates the neutralization of bacteriophage by the sera of the newborn.     

Ultrastructure and composition of bovine conglutinin.

Conglutinin binds in a Ca2+-dependent manner to the carbohydrate portion of zymosan and cell-bound iC3b (complement subcomponent C3b cleaved by Factor I in the presence of factor H) similarly to lectin-like proteins that participate in the clearance of plasma glycoproteins. This carbohydrate-binding protein has been found to include both collagenous and non-collagenous domains. Electron micrographs of bovine conglutinin are presented in which conglutinin appears as a tetramer of four 'lollipop' structures emanating from a central hub. The stem region, linking each head to the central hub, is quite stiff, whereas the hub-stem junction is a flexible hinge. From electron micrographs of a pepsin digest of conglutinin, the linkage region is identified as the collagenous portion of the macromolecule. Conglutinin is a multimer of a single polypeptide chain. From sedimentation equilibria of unreduced as compared with reduced and alkylated conglutinin, there are determined to be three disulphide-linked chains. These data, combined with information on the subunit polypeptide of conglutinin, suggest a model for conglutinin in which four disulphide-linked trimers are associated via the N-termini to form the intact macromolecule as viewed in the electron microscope. The ultrastructure of conglutinin appears ideally suited to its lectin-like function.     

The generation of chemotactic activity in sera of germ-free newborn piglets by interaction with rough strain ofEscherichia coli

The interaction of sera of newborn precolostral piglets with rough strain ofEscherichia coli or its endotoxin leads to formation of a factor which is chemotactic for rabbit polymorphonuclear neutrophil leucocytesin vitro (as tested using the Boyden's diffusion two-compartment chamber). Smooth strain ofEscherichia coli does not induce chemotaxin formation. The generation of chemotactic factor can be prevented by heating of the serum, addition of EDTA or yeast phosphomannan. The generation of the chemotactic activity is explained by the fixation of piglet complement system which is activated by rough bacteria even in the absence of detectable antibodies in newborn sera.     

The opsonic activity of complement to rough strains ofEscherichia coli in vivo in newborn precolostral pigs

The participation of complement of newborn precolostral piglets (in the absence of antibody) in the opsonization of roughEscherichia coli was investigated using blood clearance test. It was found that the complement inhibitor-yeast mannan inhibits thein vivo blood clearance when injected i. venously into newborn precolostral piglets. This inhibition takes place at the serum level and not by saturation of RES cells by mannan: sera from mannan-treated piglets are not bactericidal in anin vitro bactericidal assay against roughEscherichia coli; secondly, the presence of antibody removes the blockade of phagocytosis by mannan indicating that the RES cells are functionally capable to perform phagocytosis. On the basis of our findings we postulate that in newborn precolostral piglets in which antibody is not present in their sera, complement alone (or with cooperation with other unknown serum component) acts as an opsonin on roughEscherichia coli and is responsible for the blood clearance rate of these bacteria.     

Mannan-binding protein and conglutinin in bovine serum

Conglutinin is a bovine plasma protein which mediates the agglutination of the sensitized erythrocyte-solid phase iC3b complex (conglutination). The serum mannan-binding protein (MBP) is a lectin specific for mannose and N-acetylglucosamine. Since conglutination was shown to be inhibited specifically by N-acetylglucosamine [Leon, M.A. & Yokohari, R. (1964) Science 143, 1327-1328], the possibility was raised that conglutinin might be a bovine serum MBP. The present study, undertaken to solve this problem, revealed that bovine plasma contained an MBP besides conglutinin. These two proteins were very similar in their chemical and physicochemical properties as well as binding specificity. Both bound with high affinity (Kd = 10(-8) M) to glycoproteins terminated with mannose and/or N-acetylglucosamine residues in the presence of calcium, although conglutinin preferred N-acetylglucosamine rather than mannose. They were multimeric proteins of large molecular size (over 1,000,000 daltons, and approximately 600,000 daltons for conglutinin and MBP, respectively) and consisted of a single kind of subunit with molecular weight of around 45,000. The MBP was shown to have a collagen-like structure in the molecule, as was recently reported for conglutinin [Davis, A.E., III & Lachmann, P.J. (1984) Biochemistry 23, 2139-2144]. Despite these similarities, the MBP and conglutinin were immunochemically distinct, and the MBP did not show any conglutination activity.     

Effects of fetal versus postnatal sera upon adipose tissue stromal-vascular cells in primary culture

Summary This experiment was conducted to determine if serum factors are responsible for differences in cellularity of prenatal and postnatal pig adipose tissue as determined by in vitro measurement of cellular proliferation and enzyme-histochemical metabolic development. Cellular proliferation of stromal-vascular cells derived from rat inguinal adipose tissue was measured by [³H]-thymidine incorporation. Coverslip cultures were used for analysis of histochemical differentiation. Cells were incubated in media containing 10% fetal bovine, fetal pig, mature pig, or various combinations of these sera. Fetal bovine serum promoted more [³H]-thymidine incorporation than fetal or postnatal pig sera. Fetal pig sera also stimulated more [³H]-thymidine incorporation than mature pig sera. Sera from adult pigs promoted differentiation and lipid filling of adipocytes. Fetal pig sera stimulated histochemical expression of enzymes, but did not induce lipid filling. Fetal bovine serum produced histochemically undifferentiated cells. Addition of fetal bovine serum to media containing mature pig sera reduced lipid accumulation and histochemical reactivity of cells. This effect of fetal serum was thus due to specific inhibition of lipid deposition and not substrate restriction. These experiments demonstrated that serum factors have a major influence on morphological development of fetal and postnatal adipose tissue.     

Lysosomal enzyme activities in polymorphonuclear leukocytes,macrophages, serum,and spleen of conventional,germ-free,and antigen-free minnesota miniature swine

The activities of lysozyme, myeloperoxidase, and elastase were lower in PMNs and AMs from GF and AF Minnesota miniature piglets than in the leukocytes from their CONV counterparts. In the spleen and serum of gnotobiotic piglets only the levels of lysozyme were slightly reduced. Substantially depressed activities of these LEs were found also in PMNs from precolostral piglets in comparison with PMNs from their CONV mother. The bisassociation of GF piglets with Enterococcus liquefaciens and Escherichia coli caused an increase of LE activities in their AMs, spleens, and sera. Fewer LEs were released after phygocytic stimulation with zymosan from PMNs of GF, AF, and precolostral piglets than from PMNs of CONV animals of the same age. These data suggest that the antigenic-microbial stimulation is important for the development of normal lysosomal enzyme activities in PMNs and AMs from gnotobiotic animals.     

Cytotoxic effects of normal sera on lymphoid cells. I. Antibody-independent killing of heterologous thymocytes by guinea pig, rabbit, and human sera: role of the alternative pathway of complement activation.

The mechanisms whereby normal sera may cause the death of xenogeneic lymphoid cells in vitro have been reviewed in this study using guinea pig, rabbit and human sera as the source of activity and rat and mouse thymocytes as target cells. In all of the combinations analyzed the cytotoxic reactions were found to be mediated by complement (C) as evidenced by sensitivity of the sera towards either heat inactivation (56 °C, 30 min) or treatment with cobra venom factor or sodium ethylenediaminotetraacetate (EDTA). C activity was provided via the alternative pathway in every instance: (i) both C4-deficient guinea pig serum and C2-deficient human serum displayed cytotoxicity on the target cells; (ii) sera from all three sources were active in the absence of free Ca²⁺, which is required to activate C via the classical pathway; and (iii) GPS incubated at 50 °C for 20 min to destroy the activity of factor B of the alternative pathway lacked significant cytotoxic activity while still able to lyse sensitized sheep red blood cells, a reaction proceeding via the C142 pathway. Two independent lines of evidence appeared to exclude the possible role of antibodies in nonspecific serum cytotoxicity. First, the cytotoxic capacities and the titers of guinea pig and rabbit sera were not significantly affected after absorption with target cells in the presence of EDTA, i.e., in the absence of free divalent cations, a condition which does not interfere with antigen antibody binding. By contrast, the activity was eliminated when absorption was performed in the absence of chelating agents or in the presence of a selective Ca²⁺ chelator, sodium ethyleneglycoltetraacetate, plus excess Mg²⁺ These observations also highlight the Mg²⁺-dependence of the removal of activity by absorption. Second, γ-globulins isolated from a highly cytotoxic guinea pig serum were not toxic for rat thymocytes when tested in the presence of rat C. These results suggest that conventional antibodies, whether of “natural” origin or otherwise, are unlikely to play a role in serum-produced nonspecific cytotoxicity. Furthermore, and since incubation of human serum with rat or mouse thymocytes produced conversion of factor B, “absorption” of cytotoxic activity would seem to be more likely a consequence of the consumption of C activity via the C3 shunt than of the removal of any antibodies.     

Characterization of the bactericidal action of precolostral piglet and calf sera against gram negative enteric bacteria in rough phase

The bactericidal activities of precolostral piglet and calf sera against two Gram negative bacteria (a rough strain,Escherichia coli 16, and a non-rough strain,Shigella shigae) were studied. It was found that the bactericidal factors againstEscherichia coli 16 can be closely correlated with the complement (C′) factors which participate in the hemolysis of sheep erythrocytes sensitized with amboceptor. No specific factor againstEscherichia coli 16 could be absorbed from the serum. The bactericidal action againstShigella appeared to require other factor(s) in addition to the C′ system. This factor(s) could be removed from native or heat inactivated serum by absorption withShigella and not by absorption with other bacterial strains. Evidence is presented which suggests that this factor is not similar to the classical 19S type antibody. Results in this study indicate that the limiting factors in the bactercidal action againstEscherichia coli 16 andShigella shigae are different.     

Early antibodies to human serum albumin formed in germfree piglets

Immunization of germfree piglets with HSA at the time of birth results in formation of antibodies of IgM, IgG and IgA classes in spite of the fact that prior to immunization only IgG and IgA immunoglobulins are detectable in their sera. The possible significance of this finding is discussed. Early IgG antibodies formed in these piglets differ from late and adult pig antibodies by the presence of lower molecular weight constituents and their molecular heterogeneity corresponds to that of IgG of nonimmunized newborn precolostral piglets suggesting that small amounts of immunoglobulins formed in piglets during the intrauterine life are of antibody nature. Dedicated to Prof. F. Patočka on the occasion of his 70th birthday The work was supported by a WHO grant.     

Primary structure of bovine conglutinin, a member of the C-type animal lectin family

We report the complete amino acid sequence of bovine conglutinin obtained by structural characterization of peptides derived from the protein by various chemical and enzymatic fragmentation methods. The protein consists of 351 amino acid residues including 55 apparent Gly-X-Y repeats with two interruptions. This 171-residue-long collagenous domain separates a short noncollagenous NH2-terminal region of 25 residues from the 155-residue-long globular COOH terminus revealing the structural relation of conglutinin with mannose-binding proteins, pulmonary surfactant-associated proteins, and a complement component C1q. Eight hydroxylysine residues were found in the collagenous domain. All of these hydroxylysine residues which occupy a Y position in a Gly-X-Y triplet are possible glycosylation sites since no phenylthiohydantoin amino acid was identified in automated Edman degradation cycles corresponding to these sites. The noncollagenous COOH domain of conglutinin, on the other hand, contains a carbohydrate recognition domain which shares substantial sequence homology with C-type animal lectins. Conglutinin has the greatest sequence similarity with mannose-binding proteins and pulmonary surfactant-associated proteins.     

The role of frogs in the epidemiology of Weil's disease

Summary 1. A high percentage of frog sera (ofRana esculenta andRana fusca) reacts positively with respect to many pathogenic and apathogenic leptospira-strains; this reaction is highest (100%) with respect to the incomplete biotype ofL. icterohaemorrhagiae.2. Under natural conditions frogs do not excrete leptospirae with the urine and neither is this the case, when a single leptospira has somehow succeeded in penetrating into them. This penetration does not easily take place.3. By the inoculation of many virulent leptospirae it is possible to break the congenital immunity. Up till 3 days after the inoculation leptospirae can be detected in the blood, up till 7 days in the liver and kidneys. The frogs did not appear to fall ill.4. Frogs do not play a part in the epidemiology of Weil's disease.5. In normal frog sera precipitation of the agglutinins by means of ammonium sulphate and acetone alcohol could not be observed so definitely as in immune sera. Normal frog serum is thermostabile at 56–57°C., immune serum thermolabile at the same temperature. These differences are explained, in accordance withBordet, by the lesser strength of the antibodies in normal sera.     

Evidence that bovine conglutinin reacts with an early product of C3b degradation, and an improved conglutination assay.

When EAC43b were treated with heated serum in EDTA, reactivity with bovine conglutinin appeared rapidly, even at 0 degrees C, and almost simultaneously with the loss of C3b rosetting capacity. At the time conglutinability first appeared, there was no detectable decrease in I-A or hemolytic C3 activity, and no detectable C3 antigen release from the cells. With prolonged exposure to heated serum in EDTA, I-A (immune adherence) and hemolytic C3 activity were lost. If this exposure was at 37 degrees C, C3 antigen became strongly detectable in the supernatant fluid, and eventually conglutinability was markedly reduced or lost, whereas C3d rosettes were unaffected. We suggest that bovine conglutinin reacts with some early product of C3b degradation, rather than with C3d, and propose that this intermediate be designated C3k. We have developed a semi-quantitative assay for bovine conglutinin, utilizing a Coulter Counter to register the decrease in total particles due to red cell aggregation. By using this method, we have detected conglutination with mouse complement (C) as well as with that from man and the guinea pig.     

Isolation, cloning, and characterization of a novel phosphomannan-binding lectin from porcine serum

Mannan-binding protein (MBP) is a C-type serum lectin that is an important constituent of the innate immune defense because it activates the complement system via the lectin pathway. While the pig has been proposed to be an attractive source of xenotransplantable tissues and organs, little is known about porcine MBP. In our previous studies, phosphomannan, but not mannan, was found to be an effective inhibitor of the C1q-independent bactericidal activity of newborn piglet serum against some rough strains of Gram-negative bacteria. In contrast, the inhibitory activities of phosphomannan and mannan were very similar in the case of MBP-dependent bactericidal activity against rough strains of Escherichia coli K-12 and S-16. Based on these findings, we inferred that an MBP-like lectin with slightly or completely different carbohydrate binding specificity might exist in newborn piglet serum and be responsible for the C1q-independent bactericidal activity. Herein we report that a novel phosphomannan-binding lectin (PMBL) of 33 kDa under reducing conditions was isolated from both newborn and adult porcine serum and characterized. Porcine PMBL functionally activated the complement system via the lectin pathway triggered by binding with both phosphomannan (P-mannan) and mannan, which, unlike MBP, was effectively inhibited by mannose 6-phosphate- or galatose-containing oligosaccharides. Our observations suggest that porcine PMBL plays a critical role in the innate immune defense from the newborn stage to adult-hood, and the establishment of a newborn piglet experimental model for the innate immune system studies is a valuable step toward elucidation of the physiological function and molecular mechanism of lectin pathway.     

Bovine conglutinin is a collagen-like protein

Conglutinin is a bovine plasma protein which is relatively large and asymmetric with elevated contents of glycine and, to some extent, proline. Although its physiologic function is unknown, conglutinin is known to bind, in the presence of calcium, to yeast cell walls and to the solid-phase-inactivated third component of complement. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions, isolated conglutinin appeared to consist of a single polypeptide chain (Mr 48 000). Unreduced conglutinin consisted of a single stained band with an apparent molecular weight of approximately 300 000. Cross-linking experiments with glutaraldehyde and dimethyl suberimidate suggested that this Mr 300 000 molecule consists of six of the disulfide-linked polypeptide chains. Amino acid composition revealed hydroxylysine and hydroxyproline together with elevated glycine and proline contents. Digestion of reduced, alkylated conglutinin with bacterial collagenase resulted in formation of a precipitate which consisted of an Mr 24 000 peptide which was digested to Mr 21 000 with large quantities of collagenase. These peptides contained less glycine, proline, hydroxylysine, and hydroxyproline than did the intact protein. The supernatant from this digestion mixture was, however, enriched in these four amino acids, with glycine making up nearly one-third of the total. Prolonged digestion with pepsin at 37 degrees C resulted in an Mr 20 000 peptide which was enriched in glycine, proline, hydroxyproline, and hydroxylysine. Amino-terminal sequence analysis showed that the glycine-X-Y repeating sequence begins at residue 26.(ABSTRACT TRUNCATED AT 250 WORDS)