Demonstration in Human Plasma of a Lectin Activity Analogous to that of Bovine Conglutinin

Evidence of the existence in human plasma of an activity analogous to that of bovine conglutinin is presented. The human plasma component was characterized antigenically and functionally. Human plasma was shown to agglutinate complement-coated erythrocytes in the presence of Ca2+, and this conglutination was inhibited by EDTA. The molecule also binds to complement-reacted solid phase IgG and to zymosan in the presence of Ca2+. The binding to complement is not inhibited by N-acetyl-D-mannosamine, but is inhibited by N-acetyl-D-glucosamine, as previously shown for bovine conglutinin. Antibodies raised against bovine conglutinin cross-react with the human protein. The plasma concentration of the conglutinin-like protein showed a high inter-individual variation between apparently healthy donors. Unlike bovine conglutinin, the human conglutinin activity could not be demonstrated in serum but only in plasma. The activity was more stable in plasma containing metal-ion chelators like EDTA and citrate than in heparin or hirudin plasma.     

Characterization of a Bovine Thymic Differentiation Antigen Analogous to CD1 in the Human

Three monoclonal antibodies (MoAb), TH97A, CC13, and CC14, define a thymic differentiation antigen in cattle. The antigen is expressed on 50-60% of bovine thymocytes, located mainly in the cortical areas, but is not expressed on peripheral blood mononuclear cells (PBMC). In cryostat sections of lymph node, the antibodies react with large dendritic-like cells in the paracortical regions. They also react with a proportion of the large 'frilly' cells in afferent lymph and with dendritic-like cells in the dermis. The antibodies apparently do not react with cells in the epidermis. Biochemical analysis of the antigen recognized by MoAb TH97A reveals two bands of 44 kDa and 12 kDa under reducing conditions. These polypeptides are distinct from bovine class I major histocompatibility complex molecules reactive with the MoAb w6/32. The tissue distribution of positive cells together with results of biochemical analyses indicate that the antigen recognized by these MoAb is the bovine analogue of the human CD1.     

In Vivo and In Vitro Antibacterial Activity of Conglutinin, a Mammalian Plasma Lectin

Conglutinin is a mammalian C-type lectin which agglutinates iC3b-coated erythrocytes. Ingram [13] found that euglobulin from bovine serum may confer partial protection against experimental infections in mice. We now present evidence that the protective activity in euglobulin against infections of BALB/c mice with Salmonella typhimurium is mediated by conglutinin. Conglutinin also demonstrated antibacterial activity against E. coli and S. typhimurium in vitro. The expression of this activity required the presence of heat-labile serum factors and peritoneal exudate or spleen cells, but not antibodies to the bacteria. Antibacterial activity was also demonstrated when the bacteria were pretreated with serum at 37 degrees C before incubation with conglutinin and cells. The activity of conglutinin was not observed when factor I-deficient or EDTA-treated serum was used instead of normal serum. The active peritoneal exudate or spleen cells showed adherence to plastic.     

A Comparison of Some Properties of Bovine Conglutinin with those of Rabbit Immuno-Conglutinin

A number of properties of bovine conglutinin and rabbit immuno-conglutinin (I-K) have been compared. These are summarized in Table 1. [Table: see text]

Bovine conglutinin is seen to be an antigenically specific β₁ globulin which reacts with the alexinated complex only in the presence of calcium. A reaction with zymosan and with agar also occurs which is dependent on calcium ions but not on the presence of complement. For this reason EDTA-containing buffers should be used in immuno-diffusion studies on conglutinin. From conglutinated zymosan substantially purified preparations of conglutinin have been obtained.

Rabbit immuno-conglutinin is seen to show the properties to be expected of an antibody to fixed complement.

A preliminary account is given of an immuno-histological technique employing conglutination which will detect bound complement of various species in tissues.

By EDTA elution of conglutinated γ-globulin aggregates alexinated with guinea-pig complement a preparation of C′₁ has been obtained which had C′₁ haemolytic activity and which gives a single line in the β₁ region on immunoelectrophoresis with an anti-guinea-pig globulin serum.

     

Purification, Subunit Characterization and Ultrastructure of Three Soluble Bovine Lectins: Conglutinin, Mannose-Binding Protein and the Pentraxin Serum Amyloid P-Component

Conglutinin and mannose-binding protein (MBP) are members of the C-type lectins which are widely present in mammalian plasma. Serum amyloid P-component (SAP) is a member of the pentraxin family with lectin properties. A scheme for the partial purification of all three lectins by carbohydrate affinity chromatography and selective elution was developed. The purification was monitored by SDS-PAGE, Western blotting and electron microscopy. Binding of the lectins to Sephadex-iC3b, their collagenase sensitivity, and the size and antibody reactivity of their subunits was investigated. The demonstration, by SDS-PAGE, of 25-kDa subunits, which were unaffected by collagenase treatment but bound to Sephadex-iC3b and antibodies to human SAP, indicated the existence of bovine SAP. Bovine conglutinin (BK) also showed calcium-dependent binding to Sephadex-iC3b, whereas bovine MBP did not. The binding of BK was inhibitable with GlcNAc. A 3000-fold increase in BK activity (ELISA) was obtained in eluates from Sephadex-iC3b. SDS-PAGE analyses of BK and MBP revealed subunits with an Mr of 43 kDa and 30 kDa, respectively. These subunits were sensitive to collagenase treatment which reduced the Mr to 20 kDa. Electron micrographs revealed a prominent flexible tetramer molecule (diameter 96 nm) in the BK preparations, a predominantly hexameric structure (diameter 30 nm) in the MBP preparations, and single annular pentameric disc-like molecules (diameter 11 nm) in the SAP preparations.     

Mannan-Binding Protein and Bovine Conglutinin Mediate Enhancement of Herpes Simplex Virus Type 2 Infection in Mice

A broad range of plant lectins have recently been shown to inhibit the infectivity of herpes simplex virus type I (HSV-1) in viiro . We decided to investigate the role of mammalian Icctins in infection witb herpes simplex virus. Two lectins, conglutinin and mannan-binding protein (also called mannose-binding protein. MBP). belonging to the collectin family of lectins, were examined.
Four week-old BALB/c mice were injected subcutaneously with 100 μg bovine conglutinin or 50 μg human MBP 1 day before intravenous infection with 5 × 10⁴ PFU of herpes simplex virus type 2 (HSV-2). A three-fold increase in virus titre of the liver was observed on day 3 of the infection in the mice pretreated with conglutinin or MBP. whereas no effect was seen on days I and 5.
In a standard plaque assay using Vero cells we were not able to demonstrate reproducibly either infection inhibition or infection enhancement, when virus was pre-incubated with differing concentrations ofthe collectins. Tbe concentrations used were similar to tbose used by us in livo , and by others in in vitro experiments showing inhibition of the infectivity of HSV-1 with plant lectins.
In an ELISA with HSV-2 antigens captured on anti-HSV-2 antibodies, calcium-dependent and carbohydrate inhibitabte binding of the collectins was observed. Our results indicate that the effect of endogenous mammalian collectins in vivo may not be neutralization as suggested by the data using plant lectins. Instead, the previously described opsonizing activity of the mammalian collectins may provide the virions witb an alternative port of entry into cells leading to infection enhancement.     

Human Autoantibodies Against C1q: Lack of Cross-Reactivity with the Collectins Mannan-Binding Protein, Lung Surfactant Protein A and Bovine Conglutinin

The collectins, a group of humoral C-type lectins, have globular and collagen-like regions and share structural features with the complement protein C1q. The question was asked if autoantibodies to the collagen-like region of C1q (anti-C1qCLR) might cross-react with collectins, such as mannan-binding protein (MBP), lung surfactant protein A (SP-A) and bovine conglutinin (BK). Anti-C1qCLR antibodies of the systemic lupus erythematosus (SLE) type and anti-C1qCLR antibodies of the hypocomplementemic urticarial vasculitis syndrome (HUVS) type were investigated. Cross-absorption and elution experiments combined with antibody detection by enzyme-linked immunosorbent assay (ELISA) and immunoblot analysis gave no evidence of cross-reactive anti-C1qCLR antibodies. However, one serum with HUVS type anti-C1qCLR antibodies contained anti-MBP antibodies that were cross-reactive with SP-A. Judging from results of ELISA inhibition experiments and immunoblot analysis, four SLE sera contained antibodies to native BK, while two sera with HUVS type anti-C1qCLR antibodies contained antibodies to epitopes of denatured BK. This might imply that autoimmunity to collagen-like structures is not restricted to C1qCLR in HUVS and HUVS/SLE overlap syndromes.     

Characterization of a Monoclonal Antibody that Binds to Apolipoprotein E and to Lipoprotein of Human Plasma Containing APO E. Applications to ELISA Quantification of Plasma APO E

Abstract

This study describes the development of an enzyme-linked immunosorbent assay for human apolipoprotein E (apo E).

A mouse monoclonal IgG₁ antibody named E01 against apolipoprotein E was selected from five antibodies secreted by hybridomas. This antibody had a high affinity for apo E ((K a 1.2 × 10⁷ L.M^?1 for purified apo E and K = 1.05 × 10⁷ L.M^?1 for native apo E in very low density lipoproteins) in liquid phase and recognized every isoform of apo E but not other proteins in VLDL. Competition experiments with ¹²⁵I apo E showed that its binding affinity for the E in every density class (VLDL, HDL, LDL) and in serum was the same.

This antibody was used for the quantification of human apo E in serum by enzyme linked immunoassay. E01 was coated on microtiter plates and a polyclonal peroxidase-conjugate was used as second antibody. A good correlation was observed between the values obtained for apo E using both monoclonal and polyclonal antibodies.     

Characterization of a Galactose-Specific Lectin from Actinomyces viscosus by a Model Aggregation System

A simple model system has been developed in which lectin-mediated aggregation of glycoprotein-coated beads can be monitored by following the decrease in light scattering at 650 nm. Aggregation has been characterized with the lectin of Actinomyces viscosus T14V. Its dependence on pH, temperature, and stirring rate was examined, and the number of bacterial cells in relation to the number of latex beads resulting in optimal aggregation was established. This system has the advantage of permitting the study of a single ligand of defined structure. The ligand density was determined with radiolabeled glycoproteins. Under the conditions of the assay, ligand leakage was less than 3%, and ligands were not displaced from the beads by various proteins, glycoproteins, or by other components present in the assay mixture. Latex beads coated with asialofetuin aggregate upon the addition of A. viscosus T14V cells. By contrast, when asialofetuin was first extensively treated with purified galactose oxidase, no aggregation occurred. Only after reduction with NaBH₄ was aggregation restored, demonstrating that galactose termini of asialofetuin are essential for the binding of A. viscosus lectin. An absolute requirement for calcium was also demonstrated. Various sugars inhibited aggregation in the following order, starting with the most effective: lactose, methyl-β-D-galactopyranoside, galactose, N-acetylgalactosamine, methyl-α-D-galactopyranoside. Beads coated with fimbriae from A. viscosus coaggregated with neuraminidase-treated human erythrocytes and with Streptococcus sanguis cells. In each instance the aggregation was inhibited by lactose, indicating that the A. viscosus lectin is located in the fimbriae. Cells grown under different conditions differed in their effectiveness in aggregating glycoprotein-coated beads, suggesting differences in lectin density or accessibility. Two different experimental designs were used to establish the minimum ligand density for aggregation to occur. In one type of experiment, a threshold concentration was found for asialo α₁-acid glycoprotein, but not for asialofetuin. With an alternate approach in which a different population of galactose residues was exposed, a threshold phenomenon was also demonstrated for asialofetuin. The importance of structural ligand features in the aggregation assay is discussed in view of these findings.     

人P选择素lectin基因片段的克隆及表达

应用PCR技术扩增P选择素lectin基因 ,克隆入pET42b (+)载体 ,测序验证后在大肠杆菌BL2 1中表达了lectinC端融合 6×His蛋白 ,表达产物经Ni2 + NTAsuperflow亲和柱纯化 ,纯度可达 90 %以上 ,得率为 0 9mg/ 10 0ml,其表达量达到总菌体蛋白的 15 %。SDS PAGE和Western印迹试验显示 ,表达产物相对分子质量为 130 0 0。     

Inhibition of Human Lymphocyte Transformation by Tomato Lectin

The lectin from tomato ( Lycopersicon esculentum ) fruits was found to be non-mitogenic for human lymphocytes in culture and actually suppressed spontaneous DNA synthesis. It also inhibited the transformation of human peripheral blood Lymphocytes induced by recall antigens or allogeneic cells in vitro. This inhibition was most effective when the lectin was present from the beginning of the culture period, and could be abolished by the simultaneous addition of oligomers of N-acetylglucosamine The tomato lectin was able to bind to several major lymphocyte cell surface glycoproteins, but not to the major histocompatibility (HLA) antigens. The binding of tomato lectin to lymphocytes could be inhibited by wheal germ agglutinin (WGA). but not by concanavalin A. Tomato lectin could agglutinate monocytes and B lymphocytes as well as T lymphocytes. Human serum used to supplement the culture medium supporting lymphocyte transformation was equally effective after passage through a tomato lectin-Sepharose column. The inhibition of lymphocyte transformation brought about by tomato lectin was not stopped by exogenously added interleukin 1 and/or interleukin -2 even at very high concentrations.     

Exposure to Human and Bovine Noroviruses in a Birth Cohort in Southern India from 2002 to 2006

Human and bovine norovirus virus-like particles were used to evaluate antibodies in Indian children at ages 6 and 36 months and their mothers. Antibodies to genogroup II viruses were acquired early and were more prevalent than antibodies to genogroup I. Low levels of IgG antibodies against bovine noroviruses indicate possible zoonotic transmission.     

Identification by Full-Genome Analysis of a Bovine Rotavirus Transmitted Directly to and Causing Diarrhea in a Human Child

The genome of rotaviruses consists of 11 segments of double-stranded RNA, and each genome segment has multiple genotypes. Thus, the genotype constellation of an isolate is often indicative of its host species. Albeit rarely, interspecies transmission occurs either by virions with nonreassorted or reassorted genomic segments. A rotavirus with the G6P[1] genotype, Ro8059, was isolated from the stool of a 1-year-old child during routine characterization of diarrheal specimens from a sentinel clinic in Israel in 1995. Since genotype G6P[1] is generally associated with bovine rotaviruses, and the child developed diarrhea within days of his first contact with calves at an urban farm, the aim of this study was to characterize the whole genomic constellation of Ro8059 and four G6P[1] bovine strains, BRV101, BRV105, BRV106, and CR231/39, by RNA-RNA hybridization and full genome sequencing to determine whether some or all of the segments were of bovine origin. The genome constellations of all four bovine G6P[1] strains were G6-P[1]-I2-R2-C2-M2-A3-N2-T6-E2-H3 for VP7-VP4-VP6-VP1-VP2-VP3-NSP1-NSP2-NSP3-NSP4-NSP5, respectively. Ro8059 shared the same genotype constellation with these bovine strains, with high nucleotide sequence identities (95.84 to 100%) for each of the 11 segments indicating that Ro8059 represented a direct interspecies whole-genome transmission of a nonreassorted rotavirus from a calf to a human infant. We conclude that this was the earliest example with a complete epidemiological link in which an entirely bovine rotavirus directly infected a human child and caused a symptomatic diarrheal illness. Thus, not all bovine rotaviruses are always naturally attenuated to the human host.     

人胚食管上皮凝集素结合部位的观察

选用四种凝集素(WGA、UEA、LCA、BSA)对人胚食管采用ABC法进行石蜡切片的标记.结果发现四种凝集素对不同胚龄的食管呈不同反应.LCA在8月开始于基底层细胞出现微弱阳性反应.在3月,WGA巳能与基底层细胞膜明显结合,UEA和BSA可与非纤毛区细胞结合,而不与纤毛区细胞结合.出生时,四种凝集素的染色均未达到成人食管的染色强度.本研究结果提示,上述凝集素在人胚食管上皮的结合部位的分布情况及染色强度,可作为该上皮细胞的发育、分化程度的标志之一.     

Human Mannose-Binding Lectin Inhibitor Prevents Myocardial Injury and Arterial Thrombogenesis in a Novel Animal Model

Myocardial infarction and coagulation disorders are leading causes of disability and death in the world. An important role of the lectin complement pathway in myocardial infarction and coagulation has been demonstrated in mice genetically deficient in lectin complement pathway proteins. However, these studies are limited to comparisons between wild-type and deficient mice and lack the ability to examine reversal/inhibition of injury after disease establishment. We developed a novel mouse that expresses functional human mannose-binding lectin (MBL) 2 under the control of Mbl1 promoter. Serum MBL2 concentrations averaged approximately 3 μg/mL in MBL2^+/+Mbl1^−/−Mbl2^−/− [MBL2 knock in (KI)] mice. Serum MBL2 level in MBL2 KI mice significantly increased after 7 (8 μg/mL) or 14 (9 μg/mL) days of hyperglycemia compared to normoglycemic mice (P < 0.001). Monoclonal antibody 3F8 inhibited C3 deposition on mannan-coated plates in MBL2 KI, but not wild-type, mice. Myocardial ischemia/reperfusion in MBL2 KI mice revealed that 3F8 preserved cardiac function and decreased infarct size and fibrin deposition in a time-dependent manner. Furthermore, 3F8 prevented ferric chloride–induced occlusive arterial thrombogenesis in vivo. MBL2 KI mice represent a novel animal model that can be used to study the lectin complement pathway in acute and chronic models of human disease. Furthermore, these novel mice demonstrate the therapeutic window for MBL2 inhibition for effective treatment of disease and its complications.The innate immune system plays an important role in host defense. The complement system, as a part of the innate immune system, is involved in protection against pathogens.¹ The complement cascade can be activated/initiated through three distinct pathways: classic, alternative, and lectin. Lectin pathway (LP) activation is initiated by the presence of specific structures on microorganisms (bacterial, fungal, and some viral) binding to IgM or by changes in glycosylation patterns on compromised cells.^2–5 There are several pattern recognition molecules that may be involved in LP activation, such as mannose-binding lectin (MBL) 2 (Mbl1 and Mbl2 in mice), ficolins (1, 2, and 3), and collectin 11.^6,7 These pattern recognition molecules are associated with MBL-associated serine proteases (1, 2, and 3) and can directly activate C4 and C2 in the LP.^3,4,7–17 MBL plays a significant role as an initiation molecule that recognizes endogenous ligands after oxidative stress and tissue injury, ultimately leading to vascular wall remodeling, thrombogenesis, and other cellular injuries.^2,5,18–22Studying the role of MBL in animal models of human disease has been limited to comparison of wild-type (WT) mice to Mbl-null mice, as well as reconstitution of Mbl-null mice with human recombinant MBL2. There are no inhibitors to both murine Mbl1 and Mbl2, and this prevents the study of the therapeutic window in animal models of human disease. Furthermore, the lack of inhibitors to murine Mbl1 and Mbl2 does not allow the study of disease succession or reversal of outcomes after MBL complex activation in disease models. To circumvent these limitations and to advance the field, we generated a novel human MBL2-expressing mouse that lacks murine Mbl1 and Mbl2. We report that MBL2^+/+Mbl1^−/− Mbl2^−/− (MBL2 KI) mice produce functional human MBL2 and display LP activity similar to WT mice. Furthermore, anti-MBL2 (clone 3F8) monoclonal antibody (mAb) in the MBL2 KI mouse significantly protects the ischemic/reperfused murine myocardium from loss of myocardial function, decreases myocardial infarct size, and prevents myocardial fibrin deposition and occlusive thrombogenesis in vivo.     

Characterization of Latent Transforming Growth Factor-β From Human Seminal Plasma

PROBLEM : Human seminal plasma is known to exhibit immunosuppressive activity. Transforming growth factor β (TGF-β) has been identified as an immunosuppressive factor in human seminal plasma. Biologically active TGF-β represents a family of 25-kDa homodimeric proteins linked with disulfide bonds. TGF-β associates with high molecular weight proteins noncovalently to form a type of latency that is biologically inactive. Quantitative distribution of active form of TGF-β versus inactive latent form of TGF-β, and mechanism of the TGF-β activation in human seminal plasma remain to be elucidated. PURPOSE : To characterize seminal plasma latent form of TGF-β, including its concentration, and the mechanism underlying the activation of TGF-β. METHOD : Gel filtrations on ACA-34 and Biogel P-60 were used to fractionate seminal plasma. TGF-β was measured by enzyme immunoassay using antibodies specific for TGF-β₁ and TGF-β₂, respectively. Radioreceptor assay with recombinant human [¹²⁵I]-TGF-β₁ was applied to qualitatively identify TGF-β₁. Kinetic experiments with various pH, temperature and time, along with protease inhibitors, were performed to delineate the activation mechanism of latent TGF-β. RESULTS : Human seminal plasma contained both TGF-β₁ and TGF-β₂, predominantly in latent form. The total concentration of TGF-β₁ averaged 238 ng/ml versus an average of 18 ng/ml for TGF-β₂. The in vitro activation or release of TGF-β₁, from latent TGF-β₁ was achieved only at acidic pH of <4.0, and was time and temperature dependent. At pH 3.7 and 37°C, a significant activation of latent TGF-β₁ was achieved after an incubation of only 15 min, reached the maximum at 120 min, and the activated TGF-β₁ remained relatively stable for at least 24 h. The activation was not inhibitable by a series of protease inhibitors examined, alone or in combination (e.g., phenylmethylsulfonyl fluoride, E-64, pepstatin, leupeptin, ethylenediamine tetraacetic acid). Competitive radioreceptor assay established the functional identity of TGF-β₁ in human seminal plasma with recombinant human TGF-β₁. CONCLUSION : Human seminal plasma TGF-β is biologically activated from high molecular weight latent TGF-β by acid pH. The acidic environment of female lower genital tract could represent an in vivo physiological condition for activation of seminal plasma TGF-β that may immunologically protect the integrity of sperm.     

Identification and Characterization of a Human Sperm Antigen Corresponding to Sperm-Immobilizing Antibodies

ABSTRACT: A sperm antigen has been isolated front radiolabeled human sperm cell membrane by detergent solubilization, lectin affinity chromatography, gel filtration, and indirect immune precipitation using sperm-immobilizing antisera from patients with unexplained infertility. Isolated material was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Among 20 infertile women's sera with sperm-immobilizing antibodies, two were found to react predominantly with a sperm membrane polypeptide having the approximate molecular weight of 15,000 daltons. No significant binding to this molecule was observed in any sera from pregnant women, unmarried women, and normal men. By the absorption with spermatozoa, the antisera lost their binding activity to the molecule, while the sera absorbed with seminal plasma did not lose the activity. The results indicated that the molecule is a genuine sperm antigen and not a sperm-coating seminal plasma antigen. By the indirect immunofluorescence of washed ejaculated spermatozoa with the antisera, strong fluorescence was localized only in an equatorial segment of the acrosome, while no specific staining was observed in the controls. The antigen is relatively unstable against acid, alkali, and heat treatment. Treatment with proteolytic enzymes such as pronase and trypsin inactivated the antigen activity, indicating that the antigen epitope could be a peptide portion of the glycoprotein.     

牛骨胶原基质的制备和性能研究

研究了从牛密质骨制备用作生物医学异种移植物的加工方法。将从屠宰场获得的牛腿骨骨干中部密质骨切割成一定的形状,然后经过脱脂和脱蛋白,制得可加工成任意形状的材料。用脱脂剂、非胶原蛋白除剂和蛋白酶消化剂等进行处理,脱除非胶原蛋白并切除胶原端肽,骨块经干燥,γ射线消毒后,密封包装。并进行各种理化性能测试,包括红外光谱、X射线衍射光谱、差热扫描分析(DSC)、单轴拉伸力学性能测试以及扫描电镜观察。结果表明,经过一系列处理后,获得的骨胶原基质(BBCM)是一种白色、具有一定孔隙结构的材料,保留了天然骨的无机成分和大部分胶原蛋白,具有良好的力学性能和较低的免疫原性,是一种潜在的骨移植材料和组织工程基质材料。