Macroglobulin Fraction Research Articles

Maternal, placental and cord zinc components in healthy women with different levels of serum zinc.

Serum zinc is known to decrease during pregnancy, but the range of individual values at the end of normal gestation may be considerably large, with uncertain physiological significance in terms of maternal zinc status and maternal-fetal transfer of zinc. In this study we compared several maternal and cord blood indices of zinc status, placental zinc, placental metallothionein and the relationship between maternal, cord and placental components, in 40 healthy pregnant women at delivery with different levels of serum zinc. Subjects were divided into three groups according to serum zinc using as cutoff points the lower and upper quartile values [LZn, < 7.6 mumol/l (n = 10); MZn, 7.6-10.7 mumol/l (n = 20), and HZn, > 10.7 mumol/l (n = 10)]. Habitual zinc intakes were similar in all groups (average 11.5 mg/day). Considering all women, maternal serum and erythrocyte zinc correlated significantly (r = 0.40; p = 0.021). Maternal erythrocyte zinc was higher (p < 0.01) in HZn compared to the other groups. Maternal and cord values of serum zinc correlated significantly (r = 0.43; p = 0.006). Cord serum zinc values were very similar in LZn and MZn but were higher (p < 0.01) in HZn. Cord erythrocyte zinc levels were similar in all groups and about 20-25% of the maternal values. Cord erythrocyte metallothionein levels were also similar in all groups and similar to maternal values. Comparing the percentage distribution of zinc in maternal and cord serum fractions, major differences were observed in HZn, with zinc in albumin fraction being 70% in cord compared to 56% in maternal serum. There was a higher (p < 0.01) percentage of zinc bound to alpha 2-macroglobulin fraction in maternal serum of HZn compared to maternal serum of the other groups. Maternal and cord zinc in the albumin fractions correlated significantly (r = 0.48; p = 0.002) particularly in HZn (r = 0.71; p < 0.021). Placental zinc correlated negatively (r = -0.34; p = 0.035) with zinc in the maternal alpha 2-macroglobulin fraction, but did not relate to placental metallothionein, which had similar levels in all groups (average 0.28 nmol/g wet tissue). Placental zinc levels were lower (p < 0.01) in HZn. Our results indicate that high levels of maternal serum zinc in healthy women at delivery may be related to maternal tissue zinc redistribution that could favor diffusional components of maternal-fetal transfer of zinc.

Venom resistance in the Hedgehog, Erinaceus europaeus: Purification and identification of macroglobulin inhibitors as plasma antihemorrhagic factors

Hedgehog ( Erinaceus europaeus) plasma contains factor(s) which neutralize the hemorrhagic activity of European viper ( Vipera berus) venom. These antihemorrhagic factor(s) were purified by gel filtration on Sephacryl S-200 and chromatography on Cibacron Blue Sepharose. The macroglobulin fraction obtained was characterized for proteinase inhibiting activity, molecular weight and purity by polyacrylamide gel electrophoresis and immunological cross-reactivity and was found to contain the three macroglobulin proteinase inhibitors— α 2- macroglobulin , α 2-β- macroglobulin and β-macroglobulin. The purified macroglobulins were totally able to neutralize Vipera berus venom hemorrhagic activity, but no distinction between them in this respect was possible.

A search for antibodies in term maternal sera to solubilized syncytiotrophoblast surface components: A passive haemagglutination assay yields negative evidence

Using a passive haemagglutination assay, agglutinins against trophoblast components were detected in term maternal sera at titres above those of non-pregnant control sera. These agglutinin titres were independent of the family relationship between individual sera and trophoblast samples. They were unaffected by adsorption with excess packed fetal or paternal red blood cells or paternal lymphocytes. The agglutinins of both normal control and term maternal sera were found to elute in the macroglobulin fraction of serum having a molecular weight of > 350 kDa and were shown to be a component of euglobulin precipitates. The titres of the agglutinins were unaffected by adsorption using solid phase antibodies against human IgM and IgG from the sera. It was concluded that these agglutinins were not a maternal antibody response against trophoblast surface components.

On the role of the pancreatic secretory trypsin inhibitor as an inactivator of trypsin-alpha 2-macroglobulin complexes in acute pancreatitis.

High levels of immunoreactive pancreatic secretory trypsin inhibitor (PSTI) were demonstrated in the serum and peritoneal exudates of patients suffering from acute pancreatitis. Trypsin-like immunoreactivity in these fluids was found in complex with alpha 1-antitrypsin and in complex with alpha 2-macroglobulin and also as a free peak correlating to free trypsin(ogen). No trypsin-PSTI complexes or PSTI were demonstrated in the macroglobulin fraction of the peritoneal exudates. Saturated and partially saturated trypsin-alpha 2-macroglobulin complexes were prepared in vitro. PSTI was able to partially inhibit the BzArgNan-cleaving activity of both types of complexes in a slow dose-dependent non-linear reaction. Equilibrium was reached in each case within 1 h, but total inhibition was not reached even with large amounts of PSTI. Partially saturated trypsin-alpha 2-macroglobulin complexes were inhibited more readily than saturated complexes. The results support the concept of PSTI acting as a strictly local inhibitor of trypsin in compartments lacking plasma protease inhibitors.

Attachment of Trypanosoma brucei to rabbit peritoneal exudate cells

Attachment of trypanosomes to cultured rabbit peritoneal cells was enhanced in the presence of hyperimmune Trypanosoma brucei antiserum and sera from infected rabbits. During infection attachment rose rapidly from control levels reaching a maximum value after two or three weeks; this was maintained until the death of the animal. The initial rise in activity was preceded by an increase in the serum titres of trypanosome agglutinating antibody. Attachment did not appear to be mediated by variant specific antibodies, no association being found between adherence and the appearance of successive variant subpopulations. Fractionation of hyperimmune and immune sera indicated that the majority of activity was present in the gamma globulin fraction with less activity in the macroglobulin fraction, despite its elevation during infection. Increased activity obtained with partially-purified immunoglobulin G prepared from hyperimmune serum was reduced following absorption with either disrupted or live trypanosomes.

Polyclonal B cell activator associated with alpha-2-macroglobulin in the serum of patients with rheumatoid arthritis.

We investigated whether patients with rheumatoid arthritis have a polyclonal B cell activator (PBA) in their serum by using three methods: (1) the ability of any PBA to maintain the surface Ig of rabbit or human B cells in vitro; (2) the induction of blast transformation in human B lymphocyte cultures, and (3) stimulation of nude mouse spleen cells in vitro. All three methods indicated that a PBA is present in the serum of patients with rheumatoid arthritis but not in normal individuals or in patients with arthritis in which autoimmune phenomena have not been demonstrated. The entire PBA activity in rheumatoid arthritis patient serum was found associated with the macroglobulin fraction obtained by Sephadex G-200 chromatography and was precipitated by rabbit anti-human alpha 2-macroglobulin, but not by rabbit anti-Ig antibody. When alpha 2-macroglobulin was purified from patient serum the entire PBA activity was recovered in this fraction. Normal alpha 2-macroglobulin prepared by the same procedure had no PBA activity. Thus, the existence of a PBA associated with alpha 2-macroglobulin was demonstrated in serum of patients with rheumatoid arthritis.

Inhibition of bovine trypsin with human plasma inhibitors

Hydrolysis of TAMe (tosyl arginine methyl ester) by trypsin was not inhibited by undiluted plasma or its pseudoglobulin fraction. Dilution of plasma resulted in inhibition of trypsin activity. Inhibition of tryptic hydrolysis of TAMe by urinary trypsin inhibitor (UTI) was prevented by macroglobulin fractions of pseudoglobulin. When case in was used as the substrate a similar phenomenon was not observed because the trypsin- α 2macroglobulin complex does not hydrolyze casein. Highly purified α 2macroglobulin ( α 2M) protected trypsin against other proteinase inhibitors ( α 1antitrypsin or UTI). Mixture of 1.1 μM of α 1antitrypsin ( α 1AT) and 9.1 × 10 −2μM of α 2M did not inhibit 8.3 × 10 −2μM of trypsin significantly, but one tenth amounts of α 1AT (1.1 × 10 −1μM) and 9.1 × 10 −3μM of α 2M showed a large extent of inhibition of 8.3 × 10 −2μM of trypsin. These results may suggest that α 2M rather protected trypsin from its inactivation by other proteinase inhibitors. Such α 2M-trypsin complex still hydrolyzes molecules of certain molecular weights such as tripeptide-pNA (H-D-Val-Leu-Lys-pNA, M.W. 552).

Neutralization and Enhancement of Infectivity of Non-salmonid Fish Rhabdoviruses by Rabbit and Pike Immune Sera

SUMMARY Plaque neutralization assays were carried out using the rhabdoviruses of pike fry (2 isolates), spring viraemia of carp and grass carp. When rabbit anti-pike fry rhabdovirus sera were used, no neutralizing activity was observed but plaque counts increased with heat-inactivated sera or sera that had been stored at -20 °C for prolonged periods. Antibody binding to the virus was demonstrated by gradient centrifugation experiments. The use of immune serum alone or in combination with anti-rabbit gamma globulin serum resulted in aggregation of radioactive virus. Aggregation was also observed when anti-rabbit gamma globulin serum was added under neutralization test conditions. Addition of non-inactivated guinea-pig serum to mixtures of pike fry rhabdovirus and rabbit antiserum to spring viraemia of carp virus resulted in strong neutralization, whereas this antiserum alone or in the presence of inactivated guinea-pig serum gave an increase in plaque numbers. The increase in plaque number and the complement-dependent neutralization phenomena were weak or absent in the homologous spring viraemia of carp virus system. Immunization of pike with pike fry rhabdovirus resulted in an increase in virus neutralizing activity which could be attributed to the macroglobulin fraction of the serum. After immunizations over a period of 6 months with virus antigens, no further increase in neutralizing activity was observed. Pike sera showed 50% plaque reduction at concentrations between 0.017 and 0.3%. Using these sera it could be demonstrated that the grass carp virus and the V 75/94 isolate from pike fry were indistinguishable from the original pike fry rhabdovirus, whereas spring viraemia of carp virus was different.

Two Fractions of Macroglobulin XaI isolated by CM-cellulose Chromotography

Human citrated plasma was treated with ammonium sulfate at 30-50% saturation, and applied to the column of sephadex G-200. Macroglobulin XaI, which was obtained by gel filtration on Sephadex G-200, was separated to two fractions by CM-cellulose chromatography. One fraction (Fr. A) was eluted with pH 6.0, 0.02M acetate buffer, and another fraction (Fr. B) with 0.06M NaCl in the same buffer by 0→0.2M NaCl gradient elution on CM-cellulose. Rechromatography of Fr. A and Fr. B resulted in the same elution pattern as the first chromatography, namely it was obtained Fr. A and Fr. B from Fr. A, and Fr. B (a) and Fr. B (b) from Fr. B.These observations have given rise to the speculation that anti-Xa substance in Fr. A may be identical with Fr. B.Fr. A showed one main band and many other bands on polyacrylamide gel disc electrophoresis, and three precipitin lines besides α2-macroglobulin on immunoelectrophoresis. While, Fr. B (Fr. B (a), Fr. B (b)) with anti-Xa activity showed one main band and two other thin bands on polyacrylamide gel disc electrophoresis, but only one distinct precipitin line (α2-macroglobulin) on immunoelectrophoresis. These observations indicate that anti-Xa substance in macroglobulin fraction is α2-macroglobulin.

Inhibition for activated factor X (Xa) of Xa inhibitor in macroglobulin fraction

Activation factor X inhibitor (XaI) of macroglobulin fraction was partially purified by ammonium sulfate saturation method, gel filtration on Sephadex G-200 and CM cellulose chromatography. This XaI made good inhibition for activated factor X (Xa) at 37°C, 25°C and even 4°C. During incubation of the XaI with Xa, there was much decrease of Xa inhibiting activity of the XaI.Therefore, it was suspected that the XaI inhibited the Xa activity stoichiometrically rather than enzymatically.

Experimental studies on the antigenicity of house dust and properties of anti-house dust antibody (author's transl)

As the precipitating antibodies in rabbits to house dust were considered to correspond to the blocking antibodies produced by hyposensitization treatment, rabbit anti-house dust antibody was used to investigate the antigenicity of house dust producing blocking antibody in human. The summary of the obtained results were as follows: 1) By the use of rabbit anti-house dust serum it was found that house dust extract contained at least three antigenic elements and the specific activity of the antibodies to house dust was displayed by the 7S gammaglobulin and not by the 19S macroglobulin fraction in radioimmunodiffusion test. 2) Fractionation of the house dust extract by gel diffusion and radioimmunoelectrophoresis disclosed that the protein fraction contained two antigenic components. Furthermore, one of these components was deprived of its antigenecity by the action of pronase but no antigenicity was demonstrated in polysaccharide fraction. On the other hand, Morris and his co-workers were unable to detect significant loss in skin reactivity to purified house dust fraction submitted to treatment with pronase. 3) Using mite extract and candida extract, cross reactions to anti-house dust serum were investigated with gel diffusion, but reactions were negative for both while Miyamoto et al. reported a close correlation between extract of house dust and considered mites as the main allergen in house dust. 4) From these results, the allergenicity of house dust in skin test and the antigenicity of house dust to blocking antibodies in man might be dissociated in some instances.

Immunoglobulin class of antibodies in monkeys infected with Toxocara canis

Sera of monkeys ( Macaca sinica), experimentally infected with multiple doses of eggs of Toxocara canis, were subjected to Sephadex G-200 gel filtration and DEAE cellulose ion exchange chromatography. The distribution of antibodies in the various serum fractions was determined by the conglutinating complement absorption test (CCAT) and the agar-gel diffusion precipitin test using antigens prepared from the adult and the embryonated eggs of T. canis and by the formation of precipitates at the oral orifice of artificially hatched living 2nd stage larvae of T. canis. In serum samples taken 1 and 2 weeks after initial infection, CCAT antibodies were located mainly in the macroglobulin fractions of serum. By 1 to 4 weeks after a third dose of eggs such antibodies were located mainly in the 7S fraction of immunoglobulins. Antibodies detected by gel precipitation were located in the macroglobulin fraction of serum, while those that produced oral precipitates on 2nd stage larvae occurred in the 7S fractions.

Attempts to demonstrate "tumor-globulin" in sera of patients with malignant neoplasms.

Attempts were made to obtain “anti-tumor globulin” serum by immunization of rabbits with electrophoretically purified globulins derived from human cancer sera by the method of Tal and Halperin. Although an absorbed, mono-specific anti-serum was obtained which produced a precipitation arc with an alpha-2 macroglobulin fraction of serum from patients with cancer, the same arc was noted with normal human serum. This fraction was demonstrated immunoelectrophoretically in 50 normal and 115 human cancer sera. Thus, in our laboratory, it was not possible to demonstrate a “T-globulin” which was unique to the serum of patients with malignant neoplastic disease.

Chicken Antibodies to Group a Streptococcal Carbohydrate

Abstract Adult chickens were hyperimmunized with group A streptococcal vaccine in an attempt to elicit the formation of high levels of antibody of restricted heterogeneity. All birds produced precipitating antibody after 3 weeks of immunization. The highest concentration was ∼8.6 mg/ml. After 6 weeks of immunization the highest level was ∼13 mg/ml and even after 6 weeks most birds had both IgM and IgY. Approximately 10% of specifically purified antibody preparations contained some IgA. In each case this was associated with the macroglobulin fraction of Sephadex G-200. Of the 37 different antibody preparations examined only ∼5% (two IgM preparations) showed a clear restriction in L chain heterogeneity. These data indicate that like rabbits and sharks, chickens can produce high levels of antibody to a group A streptococcal carbohydrate with some antibody preparations showing restricted heterogeneity.

Further studies of plasminogen proactivator

A proactivator for human plasminogen was isolated from the euglobulin fraction of human plasma. Highly purified, the proactivator shows two peaks on gel filtration, through Sephadex G-200 (both peaks are in the macroglobulin fraction), or through Biogel A-50m. The proactivator forms precipitates at 4°C overnight in Tris-HCl solution. The supernatant shows one peak in a later fraction in Biogel. Concentration of proteins in this peak and its elution through Biogel A-1.5m results in one peak for protein and proactivator activity. The proactivator loses its activity in 5 M urea, suggesting that further decomposition of the molecule results in loss of its activity. The molecular weight was calculated to be 300 000 from the elution volume in Biogel A-1.5m fractionation. The proactivator migrates in the α 2-macroglobulin region in electrophoresis, but does not cross-react with anti-α 2-macroglobulin or anti-β-lipoprotein. Ultracentrifugal analyses indicate that the proactivator has a sedimentation rate of 11.7 S ( s 20, w 15.6), and is very homogeneous. Sedimentation equilibrium analyses indicated a molecular weight of 275 000 ± 25 000. The proactivator activity of the human euglobulin fraction was demonstrated, and the yield of the purified proactivator was calculated from the activity of the starting euglobulin fraction. The proactivator has no effect on the activation of human plasminogen by urokinase.

Naturally occurring human antiglobulins with specificity for E.

Human sera have been examined for antibodies with specific reactivity for gammaE using the tanned cell hemagglutination test. Cells tanned with three different gammaE myeloma proteins provided a reproducible test system. Inhibition of agglutination reactions by gammaE proteins, but not by gammaG, gammaA, gammaM, or gammaD confirmed the specificity of these reactions. 8.5% of 304 serial serum samples obtained from miscellaneous hospitalized patients showed clear-cut anti-gamma-globulins with specificity for gammaE. In most of these instances no definite clinical history of concomitant allergic disorders could be obtained. 53% of 73 patients with well-established allergic disorders (hay fever, extrinsic asthma) showed serum anti-gamma-globulins with reactivity for gammaE. Some patients studied before and after desensitization to Bermuda grass allergen showed an increase in titer or a conversion from negative to positive reactions for anti-gammaE antibodies following several month courses of progressive desensitization. Gradient and gel filtration studies indicated that anti-gammaE globulins were 19S gammaM in all instances. No clear correlation was noted between quantitative serum gammaE levels and titer of anti-gammaE antibodies.19S serum fractions with anti-gammaE antibody activity did not release histamine from normal human peripheral blood leukocytes, whereas specific rabbit anti-gammaE antisera consistently induced leukocytic histamine release. Moreover, macroglobulin fractions with anti-gammaE activity did not block allergen-specific leukocyte histamine release induced by in vitro leukocyte challenge with allergens such as Bermuda grass and leukocytes from allergic donors. In some instances 19S human serum fractions with anti-gammaE activity appeared to potentiate histamine release when incubated concomitantly with specific allergen and leukocytes from allergic individuals.

Comparison of affinity of trypsin for two α-macro-globulin fractions and for α1-antitrypsin in dog serum

Dog serum was shown to contain two trypsin-binding α-macroglobulin fractions. The relative affinity of trypsin for these macroglobulin fractions, compared with that for α 1-antitrypsin, is still higher in the dog than the corresponding relative affinity in man, and the trypsin is more firmly bound to the α-macroglobulin fractions than to α 1-antitrypsin. A rapid transfer of trypsin complexed with α 1-antitrypsin to native α-macroglobulin fractions is demonstrated.

Cryoglobulins. II. The biological and chemical properties of cryoproteins in acute post-streptococcal glomerulonephritis.

Cryoglobulins isolated from patients with acute post-streptococcal glomerulonephritis, fixed complement &lt;i&gt;in vitro &lt;/i&gt;and were associated with inflammatory changes following intradermal administration to unsensitized guinea pigs. Intravenous administration to laboratory animals was followed by clinical and morphologic changes resembling anaphylactic shock or by glomerulitis with the deposition of human IgG but no host IgC on the glomerular basement membrane and in the mesangium. Gel filtration using Sephadex G200 of cryoglobulin solution dissolved in, and eluted with, barbital buffer yielded a single component in the macroglobulin fraction. Gel filtration chromatography using dilute acid yielded 2 components; a large molecular weight component which was not identified by chemical or immunologic methods and another fraction which had the same chemical composition as normal human IgG except for a lower sialic acid content. Streptococcal antibody was not detected in either fraction and streptococcal antigens were not localized in the glomeruli of animals that developed glomerulitis. These studies show that mixed cryoglobulins isolated from patients with acute post-streptococcal glomerulonephritis exhibit properties similar to those of antigen-antibody complexes and suggest that they may have a primary or secondary role in the production of glomerular injury.

Immunologic Responses of Pacific Hagfish

Abstract Hagfish serum contains naturally-occurring agglutinins which react to different degrees with heterogenetic, but non-Forssman antigens on the erythrocytes of various vertebrates. These antibodies of generally low activity were found to reside in the macroglobulin (IgM) fraction, but became undetectable after heating serum at 56°C for 30 min. Specific production of antibodies against sheep erythrocytes was regularly inducible, given suitable tactics of immunization. Hagfish which received weight-adjusted dosages every 14 days over a 70-day period at 18° showed 23- to 25-fold rises in specific hemagglutination titers. Induced antibodies were heat stable and displayed a high degree of specificity for sheep cell antigens. Chromatographic and immunoelectrophoretic analyses showed the naturally-occurring and induced antibody activity to be limited to a major macroglobulin fraction similar to but distinguishable from the IgM of higher vertebrates. Hagfish, despite their ‘most primitive vertebrate” status, are capable of vigorous production of antibodies to at least certain cellular and soluble antigens. The extent of their immunogenetic library and immunologic memory invites further investigation.

Characteristics of the Hemagglutinin Produced During Plasmodium lophurae Malaria in Chickens

During the course of Plasmodium lophurae infections in normal chickens, there was a sharp increase in the titer of a hemagglutinin which reacted better with trypsinized than with normal erythrocytes. This hemagglutinin was a typical "cold hemagglutinin" in that it was much more active at 4 C than at 37 C, was found in the macroglobulin fraction of the serum, was eluted from erythrocytes at 37 C, and was easily destroyed by 2-mercaptoethanol reduction. The injection of allogeneic erythrocytes into chickens prior to their exposure to P. lophurae resulted in a coexistent increase in cold hemagglutinin titers and enhanced resistance to parasitemia. Since this antibody was more active against altered erythrocytes than against normal erythrocytes, it may moderate resistance to malarial infections by specifically stimulating phagocytosis of parasitized erythrocytes.