Characterization of a Lectin in Human Plasma Analogous to Bovine Conglutinin

The structural characteristics of a human plasma protein analogous to bovine conglutinin were studied. The protein was previously found to bind to complement-reacted IgG in a calcium-dependent and N-acetyl-D-glucosamine-inhibitable manner and it further shows cross-reactivity with anti-bovine conglutinin antibody. By gel permeation chromatography the conglutinin activity in human plasma was localized to fractions containing proteins of Mr at around 700,000. The conglutinin was localized by one ELISA for antigen determinants and by another for biological activity. When analysed by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) under non-reducing conditions these fractions were shown to contain proteins of about 300,000. When human conglutinin-like protein, partially purified by affinity chromatography, was analysed unreduced by SDS-PAGE followed by western blotting, the cross-reacting anti-bovine conglutinin antibody bound to a protein with an Mr of 330,000. When the sample was reduced and alkylated before electrophoresis a band of 66,000 was immunostained. The 330,000 and 66,000 proteins were shown to be collagenase sensitive. 125I-iC3b was seen to bind to the 330,000 band when incubated with western blots of partially purified human conglutinin.     

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.     

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.     

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.

     

Human Amyloid P Component: A Circulating Lectin that Modulates Immunological Responses

Amyloid P component (AP/SAP), a glycoprotein, precipitated with purified snail galactans from Helix pomatia and Ariania arbustorum in a dose-dependent manner. Radiolabelled AP hinds to human peripheral blood mononuclear cells (PBMC), erythrocytes, and cells derived from human non-T, non-B acute lymphocytic leukaemia. The AP cell binding is specific in that it is dose-dependent and can be blocked both by excess cold AP and by Helix promatia galactan. although it cannot be blocked by an equal amount of the monosaccharide galactose. In vitro studies of human PBMC immune responses demonstrated that AP inhibits PBMC proliferation responses to mycobacterial purified protein derivative and to phytohaemagglutinin and the humoral, antibody response to pokeweed mitogen. The AP-induced suppression of non-specific antibody production by human PBMC was dependent on the time at which AP was added to the culture. AP was suppressive if added in the first 48 h of the 7-day culture, and the suppression could not be reversed by washing the cells after the exposure to AP. The mechanism of AP-induced immunosuppression is still unclear, but human SAP circulates as a pair of pentameric rings, having ten identical subunits that bind to galactose polymers, and our present data suggest that AP affects the immune response through its properties as a lectin.     

Lectin Activity in Entamoeba histolytica trophozoites

A lectin (carbohydrate-binding protein) has been found in extracts of a number of axenically grown trophozoites of Entamoeba histolytica strains. The strains grown in TYI-S-33 medium (Diamond et al., Trans. R. Soc. Trop. Med. Hyg. 72: 431-432, 1978) were HK-9, 200:NIH, and HM-1:IMSS. Strain HU-1:MUSC (HSC) was grown monoxenically in the same medium. The amoebic lectin agglutinated glutaraldehyde-fixed erythrocytes. This activity was pH dependent and heat and oxidation sensitive, and was destroyed by proteolysis upon autoincubation. The relative agglutinating potency of the different strains of amoebae was investigated. Strain HSC had the highest specific activity (210 U/mg of protein), and strain HM-1 had the lowest (14 U/mg). One unit of hemagglutinating activity is defined as the amount of lectin present in 1 ml of extract which will agglutinate 1 ml of 4% erythrocytes. Upon subcellular fractionation of the lectin present in extracts of strain HK-9, two-thirds of the activity was detected in the soluble, nonsedimentable (100,000 × g, 60 min) fraction. Partial hydrolysate of chitin was found to inhibit the hemagglutinating activity. Among the oligosaccharides of N-acetylglucosamine, the trimer and tetramer were the most potent inhibitors. The lectin was purified approximately 300-fold by a one-step affinity chromatography on a chitin column. The loading and elution from the column were based on the pH dependence of the lectin activity.     

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.     

Human and Porcine Pancreatic Elastases: The Comparative Enzymatic Activity with Human and Bovine Elastins

Elastase from human pancreas was purified by ion-exchange and affinity chromatography with an 85% yield and to approximately 95% homogenicity as judged by discontinuous slab-gel electrophoresis. The human enzyme differed in chemical and enzymatic properties in comparison to porcine pancreatic elastase. Human elastase antiserum exhibited no cross-reactivity with porcine elastase, but precipitated the human enzyme. The human elastase possessed 25% of the enzymatic activity of porcine elastase in solubilizing alkaline-extracted bovine elastin, but solubilized human aortic elastin at a much higher rate. The results initially suggested a substrate specificity by human elastase for human elastin, but additional data indicated that the method of purification of elastins significantly affected the rates of solubilization by both human and porcine pancreatic elastase.     

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.     

Modulatory Effects of Autologous Plasma on Functional Activity of Human Immunocompetent Cells

Blast transformation of peripheral blood lymphocytes stimulated with phytohemagglutinin and concanavalin A was studied in children with pyelonephritis and glomerulonephritis. Activity of natural killer cells from children with pyelonephritis was estimated before and after treatment with 50% autologous plasma. The autologous plasma modulated blast transformation of lymphocytes and activity of natural killer cells, which depended on the stage of diseases.     

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。     

凝固时间法测定血浆AT-Ⅲ活性

目的介绍凝固时间法测定血浆AT-Ⅲ活性的实验方法。方法将校正血浆用含肝素缓冲液稀释成不同浓度(即不同活性AT-Ⅲ),加入纯品纤维蛋白原共同孵育后,测定其凝固时间;根据不同AT-Ⅲ活性与相应凝固时间制作标准曲线;测定待检血浆凝固时间,查标准曲线即可得到待检血浆AT-Ⅲ活性。结果该法AT-Ⅲ活性在25%～125%范围内线性良好;日内变异系数为3.2%,日间变异系数为4.9%;凝固时间法与发色底物法比较相关性良好(r=0.999,P<0.05);检测178例健康人血浆AT-Ⅲ活性为105.4%±18.8%。结论该方法重复性好,结果可靠,简便实用,可用于常规检验。     

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.     

In Vitro Enhancement of AA-Degrading Activity in Human Plasma with the Plasminogen Activator Streptokinase

Radiolabelled protein AA was coupled to cyanogen-bromide-activated Sepharose 6 MB and used as a substrate to determine the AA-degrading activity of enzymes in solution. The applicability of the substrate was tested with an elastase preparation known to have AA-degrading activity. The substrate was used to determine the AA-degrading activity in fractions of normal human serum in the presence and absence of the plasminogen activator streptokinase. The AA-degrading activity was increased in fractions in which streptokinase-induced plasminogen activation had occurred. The increase in activity could be inhibited with antibodies to plasminogen. AA-degrading activity could also be increased in whole human plasma by the addition of streptokinase.     

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.     

Ulex Europaeus 1 Lectin for Demonstration of Lymphovascular Involvement in Microinvasive Carcinoma of the Cervix

Abstract

Lymphovascular invasion in microinvasive carcinoma of cervix may be an important prognostic indicator. However, a variety of tissue artifacts can make recognition of this invasion difficult in hematoxylin and eosin (H & E) sections. Ulex europaeus agglutinin I lectin (UEA I) used in an immunoperoxidasen technique as an endothelial cell marker facilitates the diagnosis. In this study, 32 cases with suspected vascular involvement were reacted with UEA I on both freshly cut paraffin sections and destained H & E slides. When blocks were available, adjacent sections were also studied with factor VIII antibody. Although there were problems with interpretation in some cases, there was good agreement between what was regarded as vascular space with routine staining and what was revealed with UEA I. Factor VIII was unhelpful. We concluded that UEA I immunohistochemistry is a useful adjunct for assessing capillary-like space involvement in microinvasive carcinoma of cervix. (The J Histotechnol 16:175, 1993)     

Demonstration of a Complement-Dependent Migration Inhibitory Activity in Normal Guinea Pig Serum

A cell migration inhibitory effect was evidentiated in normal guinea pig serum as compared with heat inactivated serum, Granuloeytes when used as target showed a greater sensitivity to this effect than lymphomonocytes or macrophages. The migration inhibitory activity of GPS is abrogated or decreased by using complement destroying agents such as: heating at 56°C for as little as 5 min, absorption on immune complexes in presence of calcium, on zymosan, on Sephadex G-50 or by adding EDTA or heparin to culture medium. The GPS dialysation fractions while exhibiting neither complement haemolytic effect nor migration inhibitory activity when tested alone, restored these functions by recombination. Absorption of GPS on homologous blood cells abrogated the migration inhibitory effect but retained the complement haemolytic function. When GPS absorbed on homologous blood cells was mixed 1:5 with heat-inactivated serum (5min at 56°G), the migration inhibitory activity was regained, suggesting that the complement factors from the first sample were necessary for manifestation for the migration inhibitory activity from the heat-inactivated serum.     

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.