Rapid and Efficient Purification of Functional Collectin-12 and Its Opsonic Activity against Fungal Pathogens.

Collectin-12 (collectin placenta 1, CL-P1, or CL-12) is a newly identified pattern recognition molecule of the innate immune system. Recent evidences show that CL-12 plays important roles not only in innate immune protection against certain clinically important pathogens but also in scavenging of host molecules, leukocyte recruitment, and cancer metastasis. Furthermore, CL-12 has been shown to be associated with the pathogenesis of human diseases such as Alzheimer's disease and multiple sclerosis lesion development. Therefore, the functional consequence of CL-12 remains intriguing and awaits further elucidation. However, available protocols for the purification of recombinant CL-12 with high purity are laborious and inefficient and hamper further functional studies. Here, we report a simple, rapid, and efficient solution to obtain biologically active CL-12 with high purity. We established stable transfected Flp-In™-CHO cells expressing the recombinant CL-12 extracellular domain in high amounts. Recombinant CL-12 was purified from cell culture supernatants using a 3-step rapid purification procedure utilizing disposable affinity and ion exchange minicolumns. Purified recombinant CL-12 adopted an oligomeric structure with monomers, dimers, and trimers and retained its binding capacity towards the A. fumigatus strain that has been described before. Furthermore, we demonstrated the opsonic properties towards eight clinical isolates of A. fumigatus strains and diverse clinically important fungal pathogens. Purified recombinant CL-12 revealed a differential binding capacity towards selected fungal pathogens in vitro. In conclusion, we demonstrate a rapid and efficient purification solution for further biochemical and functional characterization of CL-12 and reveal opsonic properties of CL-12 towards diverse fungal pathogens.

Treatment potential of pathogen-reactive antibodies sequentially purified from pooled human immunoglobulin.

OBJECTIVE: Intravenous immune globulin (IVIG), pooled from human blood, is a polyspecific antibody preparation that inhibits the super-antigenic proteins associated with streptococcal and staphylococcal toxic shock, and the Shiga toxin. In addition to this toxin-neutralising activity, IVIG contains other pathogen-reactive antibodies that may confer additional therapeutic benefits. We sought to determine if pathogen-reactive antibodies that promote opsonophagocytosis of different organisms can be sequentially affinity-purified from one IVIG preparation. RESULTS: Antibodies that recognise cell wall antigens of Streptococcus pyogenes, Staphylococcus aureus, and vancomycin-resistant enterococcus (VRE) were sequentially affinity-purified from a single preparation of commercial IVIG and opsonophagocytic activity was assessed using a flow cytometry assay of neutrophil uptake. Non-specific IgG-binding proteins were removed from the S. aureus preparations using an immobilised Fc fragment column, produced using IVIG cleaved with the Immunoglobulin G-degrading enzyme of S. pyogenes (IdeS). Affinity-purified anti-S. aureus and anti-VRE immunoglobulin promoted significantly higher levels of opsonophagocytic uptake by human neutrophils than IVIG when identical total antibody concentrations were compared, confirming activity previously shown for affinity-purified anti-S. pyogenes immunoglobulin. The opsonophagocytic activities of anti-S. pyogenes, anti-S. aureus, and anti-VRE antibodies that were sequentially purified from a single IVIG preparation were undiminished compared to antibodies purified from previously unused IVIG.

Antibodies Against Sporothrix schenckii Enhance TNF-α Production and Killing by Macrophages.

Sporotrichosis is a chronic granulomatous mycosis caused by the dimorphic fungus Sporothrix schenckii. The immunological mechanisms involved in the prevention and control of sporotrichosis suggest that cell-mediated immunity plays an important role in protecting the host against S. schenckii. Nonetheless, recent data strongly support the existence of protective Abs against this pathogenic fungus. In a previous study, we showed that passive Ab therapy led to a significant reduction in the number of colony forming unit in the organs of mice when the MAb was injected before and during S. schenckii infection. The ability of opsonization to enhance macrophage damage to S. schenckii and subsequent cytokine production was investigated in this work. Here we show that the fungicidal characteristics of macrophages are increased when the fungus is phagocytosed in the presence of inactivated serum from mice infected with S. schenckii or mAb anti-gp70. Additionally, we show an increase in the levels of pro-inflammatory cytokines such as TNF-α and IL-1ß. This study provides additional support for the importance of antibodies in protecting against S. schenckii and concludes that opsonization is an important process to increase TNF-α production and fungus killing by macrophages in experimental sporotrichosis.

Lipovitellin is a non-self recognition receptor with opsonic activity.

Lipovitellin (Lv), a glycolipoprotein, is a major component of the egg yolk, which is usually regarded as an energy reserve of nutrients essential for growth and development. We have purified Lv from ovulated eggs of the rosy barb Puntius conchonius by two-step chromatography and characterized it by staining with periodic acid/Schiff reagent and Sudan black B, amino acid composition analysis, and peptide mass fingerprinting. The results of ligand and bacterial binding assays, an enzyme-linked immunosorbent assay (ELISA), and the phagocytosis test revealed, for the first time, that the purified native form of P. conchonius Lv acts as a pattern recognition molecule with multiple specificities capable of identifying pathogen-associated molecular patterns (PAMPs), including those of lipopolysaccharide, lipoteichoic acid, and peptidoglycan, rather than self components and that it can bind Gram-negative and -positive bacteria, such as Escherichia coli and Staphylococcus aureus. These tests also showed that the P. conchonius Lv functions as an opsonin capable of enhancing macrophage phagocytosis. Taken together, these characteristics suggest that in developing embryos/larvae of P. conchonius, the native form of Lv may be physiologically involved in the sensing of invading pathogens via interaction with PAMPs and in the recruitment of the primitive macrophages that appear in early embryos to phagocytose and digest the pathogens, thereby protecting them from pathogenic attacks.

A pathogenic parasite interferes with phagocytosis of insect immunocompetent cells.

Phagocytosis activity of hemocytes of the host Galleria mellonella (Lepidoptera) was modulated by the infection of the entomopathogenic nematode Steinernema feltiae (Rahbditida) and was found to be correlated with the opsonization of bacteria by hemolymph factors. The presence of nematodes resulted in a significative decrease in phagocytosis of bacteria by host hemocytes, both in in vivo and in in vitro assays. Host interacting proteins (HIPs), which appear to function as opsonic factors and are essential to perform immune responses, were removed by S. feltiae from host hemolymph, by means of its epicuticle binding properties. Host humoral factors sequestered by the parasite have been identified by monodimensional and 2D electrophoretic analysis. The data suggest that S. feltiae, living in association with symbiontic bacteria (Xenorhabdus nematophilus), develop an immune suppressive strategy to support its bacteria, which diminished the effectiveness of immunological surveillance by the host.

Humoral opsonins of the tunicate, Pyura stolonifera.

This study characterizes humoral opsonins from the tunicate, Pyura stolonifera. The predominant opsonic components in P. stolonifera hemolymph were found to be calcium-dependent lectins with broad carbohydrate specificities. The opsonic lectins were purified by carbohydrate affinity chromatography which eluted a complex pattern of proteins ranging in molecular mass from 80 to >200kDa. Reducing and two dimensional SDS-PAGE indicated that the diversity of mature lectins evident under non-reducing conditions resulted from the differential oligomerization of two polypeptide sub-units (35 and 22kDa). In addition to lectin-mediated opsonic activity, hemolymph was also found to contain proteolytically activated opsonins. These data suggest that multiple, possibly interactive opsonic systems co-exist in P. stolonifera.

Purification and characterization of a humoral opsonin, with specificity for D-galactose, in the colonial ascidian Botryllus schlosseri.

A humoral agglutinin from the hemolysate of the colonial ascidian Botryllus schlosseri was purified by affinity chromatography. This agglutinin does not require metal cations for its activity and is specific for derivatives of D-galactose. On SDS-PAGE analysis, it was resolved in two bands, of 17 and 19 kDa in reducing conditions and 15 and 16 kDa in non-reducing conditions. This behavior is due to the establishment of disulfide bridge between the thiols of cysteine, well represented in the molecule as revealed by amino acid analysis. The latter also indicated high percentages of hydrophilic residues, probably involved in sugar recognition. The lectin is an opsonin, as it increases both the phagocytic index and the number of phagocytized yeast cells. The hypothesis that this Botryllus agglutinin belongs to the galectin family of lectins is discussed.

Opsonic complement component C3 in the solitary ascidian, Halocynthia roretzi.

The recent identification of two mannose-binding lectin-associated serine protease clones from Halocynthia roretzi, an ascidian, suggested the presence of a complement system in urochordates. To elucidate the structure and function of this possibly primitive complement system, we have isolated cDNA clones for ascidian C3 (AsC3) and purified AsC3 protein from body fluid. The deduced primary structure of AsC3 shows overall similarity to mammalian C3, including a typical thioester site with the His residue required for nucleophilic activation of the thioester. AsC3 has a two-subunit chain structure, and the alpha-chain is cleaved at a specific site near to the N terminus upon activation. Ascidian body fluid contains an opsonic activity which enhances phagocytosis of yeast by ascidian blood cells, and Ab against AsC3 inhibits this opsonic activity. These results indicate that the complement system played a pivotal role in innate immunity by enhancing phagocytosis before the emergence of the vertebrates and well ahead of the establishment of adaptive immunity, which is believed to have occurred at about the time of the appearance of cartilaginous fish.

Opsonic activity of cell adhesion proteins and beta-1,3-glucan binding proteins from two crustaceans.

A beta-1,3-glucan binding protein (beta GBP) from the shore crab Carcinus maenas was purified from plasma by precipitation of the protein at low ionic strength. The protein had a molecular mass of 110 kDa, and was shown to affinity precipitate with laminarin, a soluble beta-1,3-glucan, and to cross-react with an antiserum directed toward beta GBP from the crayfish Pacifastacus leniusculus. Also, a protein from the haemocytes of C. maenas with a molecular mass of 80 kDa was found to mediate cell attachment and cause degranulation of crab cells, similar to the 76 kDa protein present in the haemocytes of P. leniusculus. Antibodies against the crayfish 76-kDa protein reacted with the crab 80-kDa protein present in the granular cells. No 80-kDa protein could be found in the hyaline cells. Using a method with FITC-conjugated yeast particles in a phagocytosis assay, both the beta GBP and the 80-kDa protein from C. maenas were shown to have opsonic activity as had beta GBP and 76-kDa protein from P. leniusculus, resulting in higher levels of phagocytosis by the crab hyaline cells. Treatment of the yeast particles with beta GBP previously reacted with laminarin (beta GBP-L) only resulted in a minor increase of phagocytosis. Moreover, if the phagocytic cells were preincubated with beta GBP-L or with the 80-kDa protein, the enhancement of the phagocytic activity by beta GBP or the 80-kDa protein were abolished, indicating that a saturable number of one kind of cell surface receptor seem to be involved in phagocytosis.

Purification and characterization of a humoral opsonin from the solitary urochordate Styela clava.

1. We have previously identified opsonic activity in the plasma of the solitary urochordate, Styela clava. 2. Here, we report the purification and further characterization of the opsonic molecule. 3. Two purification methods were employed. 4. Gel filtration yielded one strongly opsonic fraction that contained a single, electrophoretically-resolved protein. 5. Opsonic activity was dose-dependent and sensitive to tryptic digestion and heat denaturation. 6. SDS-PAGE and calibrated gel filtration indicated the opsonic protein was a 17.5 kDa monomer while isoelectrofocusing indicated a single pI of 7.0. 7. In an alternative procedure, a similar opsonic activity and protein were isolated by affinity purification using whole yeast cells.

Antibacterial effect of bovine milk antibody against Escherichia coli in a mouse indigenous infection model.

A skim-milk fraction and a whey-protein concentrate (WPC) fraction were prepared from the cows that had been immunized with E. coli isolated from the mouse intestine. The antibacterial effect of these fractions against E. coli was examined. They contained antibody with a high affinity for E. coli strain 48, a representative strain in the mouse intestine, which is composed of a large amount of IgG and smaller amounts of IgA and IgM. Although these fractions showed no bactericidal or bacteriostatic activity against E. coli 48 directly in vitro, they exhibited strong agglutination and opsonization activities against the bacteria in vitro. The bacteria opsonized with the WPC fraction were taken up more effectively by liver macrophages in vivo, compared with unopsonized E. coli, after an intravenous injection into mice. Oral administration of the skim-milk fraction to mice significantly reduced the susceptibility to the lethal toxicity of 5-fluorouracil (5 FU). The increase in the population levels of E. coli in the intestinal tract after administration of 5 FU was inhibited by oral administration of the skim-milk fraction. These results strongly suggest that specific antibody may be effective in the prophylaxis against the indigenous infection with gram-negative bacteria such as E. coli after a period of chemotherapy in cancer patients.

Identification and characterization of opsonic fibronectin in synovial fluids of patients with active rheumatoid arthritis.

A cofactor that selectively opsonizes particulate activators of the human alternative complement pathway and enhances their phagocytosis by human monocytes was identified in synovial fluids of patients with rheumatoid arthritis. The active material was present in fluids treated with protease inhibitors, was heat stable, and was unaffected by incubation with hyaluronidase. Chromatographic isolation of synovial fluid fibronectin by gelatin affinity and by immunoaffinity on antifibronectin monoclonal antibody BD4 yielded similar quantities of protein for each of 3 fluids. Synovial fluid proteins with the BD4 fibronectin epitope accounted for essentially all of the phagocytosis-enhancing activity and expressed this activity by opsonizing target activators. Additional chromatographic analyses of synovial fluid fibronectin with the BD4 epitope were carried out using Sepharose-bearing gelatin and 4 additional antifibronectin monoclonal antibodies. The opsonic materials were characterized as having 2 distinct fibronectin epitopes, which always mapped from the cell adhesive domain to the carboxyl-terminus of plasma fibronectin, but only rarely contained the gelatin binding domain.

Activation of complement by Treponema denticola.

Oral spirochetes have been shown to be associated with periodontal diseases and are present in increased numbers in lesions of greater severity. In this study, the interaction of Treponema denticola with human complement, a major antibacterial defense system, was examined. For each of two strains of T. denticola, it was found that both the classical and alternative pathways of human complement were activated in human serum upon incubation at 37 degrees C. C3 fragments were deposited on the surface of this organism following complement activation; the fragments bound included both of the major C3-derived opsonic fragments C3b and iC3b. Under incubation conditions identical to those carried out for complement activation in serum, T. denticola failed to degrade purified, hemolytically-active C3, although it readily degraded inactivated C3. Thus, despite the documented proteolytic activity of this organism, complement activation and deposition of complement-derived opsonins may be important defense mechanisms in the control of infections with T. denticola.

Immunorecognition in the freshwater bivalve, Corbicula fluminea. I. Electrophoretic and immunologic analyses of opsonic plasma components.

Plasma (cell-free hemolymph) from the freshwater clam, Corbicula fluminea, has been found to possess opsonizing activity which could be completely eliminated by preabsorption of plasma with aldehyde-fixed rabbit red blood cells (RRBC). The detergents, SDS and Chapso, consistently dissociated several major proteins (Mr 20, 40, 66 and over 200 kd) from opsonized RRBC. Moreover, a partial recovery of opsonizing activity was attributed to this pool of desorbed proteins after removal of detergent by dialysis. Some of these proteins failed to bind to RRBC if the plasma was pretreated with heat, EGTA or preabsorbed with RRBC. These treatments were also found to eliminate plasma-opsonizing activity. A monoclonal antibody raised against opsonized RRBC bound to several of the same proteins seen in SDS-PAGE and could block opsonin-mediated RRBC phagocytosis by clam hemocytes. The blocking antibody also was crossreactive with epitopes expressed at the surface of washed hemocytes. Taken together these results suggest that the candidate opsonin(s) is a divalent cation-dependent, heat sensitive protein(s) with an Mr ranging from 20 to 66 kd (under reducing conditions). Furthermore, antigenically related forms of the molecules are found both in the plasma and associated with the surface membrane of circulating hemocytes.

Immunorecognition in the freshwater bivalve, Corbicula fluminea. II. Isolation and characterization of a plasma opsonin with hemagglutinating activity.

Opsonizing and agglutinating activities of plasma from the freshwater clam, Corbicula fluminea, were found to be inhibited by the sugars, 2-deoxy-D-glucose (deoxy-Glu) and N-acetyl-D-galactosamine (GalNAc). The plasma opsonin/agglutinin was subsequently isolated by a two-step separation procedure. Aldehyde-fixed rabbit erythrocytes (RRBC) were used as a solid-phase plasma opsonin affinity absorbant, and deoxyGlu and GalNAc were used in the eluting buffer to desorb several RRBC-binding plasma proteins. The second step involved the further separation of sugar-eluted proteins by Sephacryl S-200 gel filtration. A plasma protein with an apparent molecular weight of 40 kd on SDS-PAGE under nonreducing conditions was found to possess both agglutinating and opsonizing activities. It was further shown to be composed of two identical 20 kd subunits associated through disulfide linkage(s). Although this protein shares some structural similarity with other bivalve opsonins, differences in native molecular size or subunit structure, agglutinating properties and/or sugar binding specificity support the current hypothesis that naturally occurring plasma opsonins of molluscs represent a heterogeneous group of proteins unified primarily through their lectin-like characteristic of binding specific carbohydrate determinants.

High dose intravenous immunoglobulin therapy in burn patients: pharmacokinetics and effects on microbial opsonization and phagocytosis.

Depressed serum immunoglobulin levels following severe burns may lead to subsequent infectious complications following such injuries. In a randomized study we administered multiple doses of Sandoglobulin (500 mg/kg) or albumin intravenously to patients with severe burn injuries and closely monitored serum IgG levels. Patients who received IgG therapy had earlier return of normal serum IgG levels compared to control patients; however, control patients attained normal IgG levels during the second postburn week. Serum half-lives of IgG following infusions were remarkably short (means, 47 hours for infusions within 3 days of injury and 154 hours for infusions in the third postburn week); Sandoglobulin has been reported to have approximately a 21-day half-life in normal individuals. We also measured the opsonic capacity of postburn serum, using fluorescein-labeled microbes and flow cytometry; we identified postburn opsonic defects with certain of the organisms as late as 15 days postinjury, even though serum IgG levels had normalized. These defects were corrected by the in vitro addition of Sandoglobulin to the incubation mixture.

Conformational changes and loss of opsonic function in frozen or heat-treated plasma fibronectin.

Using human peripheral blood monocytes to assay opsonic protein activity, we have examined the efficiency of various procedures for isolating fibronectin from plasma for experimental or therapeutic use. In addition, we have assessed the protein's opsonic activity after cold storage, and after heat treatment to inactivate hepatitis virus. The purification procedures recovered only 30% of available plasma fibronectin whilst cold storage and heat treatment of the purified protein removed all of its remaining opsonic activity. This was associated with no alteration in overall molecular weight or in subunit size but was accompanied by changes in ultraviolet spectrum, suggesting a conformational change in the protein structure. Initial experiments to protect the purified protein against these changes were unsuccessful and unless further attempts are more encouraging, fresh-frozen plasma may be the only current economic source of opsonically active fibronectin. Since this would waste other valuable proteins required for other purposes, the widespread use of plasma fibronectin outside of clinical trials may be unjustified at this present time.

Complement-like activity in sea urchin coelomic fluid.

A complement-like activity in echinoid coelomic cell-free fluid is described. The activity is detected by the lytic action on rabbit erythrocytes (RRBC), and by the opsonic effect on echinoid coelomic phagocytes and mouse peritoneal macrophages. This activity is very heat-labile, being completely destroyed at 37 degrees C 1/2 hr, and is inhibited by Ca2+ concentrations below 10 mM and by low pH. Lysis was complete within 3-4 min, and the titer (10(7) RRBC/ml) was 20-60 between animals. Various substances known to inhibit human complement also inhibited the lytic and opsonic activities in echinoid fluid. RRBC opsonized with echinoid fluid were attached to mouse macrophages without being internalized, an effect which resemble complement opsonization. It is concluded that an activity is present in echinoid coelomic fluid, which strongly resembles mammalian complement activated via the alternative pathway. A lectin-like activity with specificity for D-fucose was detected by the agglutination of RRBC (titer 300-600). Hemagglutination was inhibited by sugars which did not inhibit the lytic and opsonic process. On the other hand, hemagglutination was resistant to various physio-chemical treatments which led to inactivation of the complement-like system; thus these two activities seem to be unrelated. The lectin-like activity did not mediate any opsonic effect.