Problems posed by the preparation of specific anti-cytomegalovirus immunoglobulins

The authors describe the preparation of a first batch of intravenous cytomegalovirus (CMV) immune globulin at the Nancy Regional blood transfusion centre. Immune plasmas were selected from 3 640 healthy volunteer blood donors on the basis of CF antibody titers to CMV (Kolmer's method modified) of, at least, 1:8; plasmas from approximately 10% of the donors were therefore selected. The 68 liters of pooled immune plasma had à CF antibody titer of 1:16 (CMV antibody titers of 1: 10 000 and 1: 640 when tested in the ELISA assay and passive hemagglutination assay respectively). Intravenous immune globulin was produced from pooled plasma by Cohn fractionation and treatment with pepsin at pH 4; 4.8 liters of immune globulin were prepared and divided in 96 doses of 50 ml each. The final product was found to have a CMV antibody titer of 1: 32 (CF) 1: 50 000 (ELISA) or 1: 2 560 (passive hemagglutination). Recent reports on the preparation of CMV immune globulin are briefly reviewed.     

Plasma protein regulation of platelet function and metabolism

Summary This reviews summarizes our evidence suggesting that the plasma protein environment influences platelet aggregation potential and metabolic activity.Cationic proteins are capable of restoring the aggregation potential of washed human platelets. The aggregation restoring effect of gamma globulin is inhibited by more anionic proteins in subfractions of Cohn fraction IV and fractions V and VI. Artificial enhancement of the net negative charge of plasma proteins through acylation produces derivatives capable of inhibiting platelet aggregation in platelet rich plasma.The oxygen consumption of washed human platelets is lower than in platelet rich plasma while the lactate production is identical. Autologous plasma, albumin or IgG immunoglobulin restores the oxygen consumption of washed platelets to values comparable to those obtained for platelet rich plasma, while the lactate production is unaffected. Fibrinogen or IgA myeloma protein increases the lactate production, but not the oxygen consumption. Cyclic AMP levels are considerably lower in washed platelets than in platelet rich plasma. Gamma globulin and albumin causes a further decrease, which is progressive with time. Fibrinogen causes no change in platelet cyclic AMP content.It is suggested that these observations may in part be explained by the equilibrium between anionic and cationic proteins in the platelet microenvironment.This hypothesis appears applicable in certain clinical situations.     

The effect of human gamma globulin on tbe production of antibodies to the surface antigen of the hepatitis B virus in an experiment on animals

The biological assay for the determination of HBsAg in gamma globulin is proposed. The assay is based on the immunizing action of immune complexes and some specific features of secondary immune response. For this purpose, the highly purified preparation of HBsAg is injected into guinea pigs. Subsequently the animals receive human gamma globulin. An increase in the titer of anti-HBsAg antibodies in the animals preimmunized with the antigen is indicative of the presence HBsAg in human gamma globulin. In accordance with another variant of this assay, human gamma globulin is introduced into mice and 21 days later the animals are immunized with HBsAg. The appearance of anti-HBsAg antibodies in the animals, previously treated with gamma globulin, on day 4 after the injection of the antigen indicate that the animals experience the second penetration of HBsAg, i. e. its presence in previously injected human gamma globulin.     

Effect of specific antibodies on active immunity development in those inoculated with an antirabies vaccine

The study of the effect of gamma globulin introduced in different doses (0.5 and 0.25 ml/mg) in combination with Fermi rabies vaccine (observations on humans were made) and with cerebral rabies vaccine inactivated by UV irradiation (in animal experiments) demonstrated that the injection of the higher doses of gamma globulin resulted in lower geometrical mean of antibody titers. Therefore, in combined administration of rabies vaccine and gamma globulin for postexposure rabies prevention it is advisable to reduce the dose of gamma globulin by one-half.     

Isolation and characterization of the testosterone-estradiol-binding globulin from human plasma. Use of a novel affinity column.

This report concerns the purification and characterization of the testosterone-estradiol-binding globulin (TeBG) from human plasma. Cohn fraction IV was submitted sequentially to ammonium sulfate preciptation, affinity chromatography, gel filtration, and isoelectric focusing. The final product was homogeneous in polyacrylamide gel electrophoresis and sodium dodecyl sulfate gel electrophoresis. Its activity was demonstrated by the finding of slightly more than one binding site/mole for dihydrotestosterone. Association constants (M-1) at 4 and 37degreesC were ascertained for three steroids: dihydrotestosterone; 2.4 x 10(9) and 0.99 x 10(9); testosterone, 1.1 x 10(9) and 0.35 x 10(9); estradiol, 0.60 x 10(9) and 0.22 x 10(9). TeBG is a glycoprotein having a molecular weight of 94000 and both the amino acid and carbohydrate content are presented along with other physical properties.     

Specificity of peroxidase-labeled antibodies to rabbit, mouse and human immunoglobulins

The comparative study of different types of conjugates (antirabbit, antimouse and antihuman) has shown that gamma globulin fractions actively interact not only with homologous peroxidase-labelled antibodies, but also with heterologous ones. Cross reactions were most pronounced between antimouse conjugates and the preparations of human gamma globulin and, vice versa, between antihuman conjugate and mouse gamma globulin. The study has shown that the main cause of cross reactions is the presence of common antigenic determinants in the preparations of mouse and human gamma globulin.     

Effect of multiple gamma globulin administration on the effectiveness of seroprophylaxis in hepatitis A

The data provided by immunological and epidemiological studies carried out to determine the influence of the multiple injections of 10% commercial gamma globulin on the level of antigamma globulin formation and, in this connection, on the effectiveness of the prophylaxis of hepatitis A are discussed. A sharp increase in the titers of antigamma globulin in persons receiving multiple gamma globulin injections is shown. The increased amount of antibodies to gamma globulin, appearing as the result of the multiple use of this preparation, decreases the effectiveness of the seroprophylaxis of hepatitis A.     

Serum Protein Electrophoresis in Tuberculosis

Serial serum protein electrophoretic determinations at two-month intervals were carried out on 84 tuberculous patients undergoing treatment in a sanatorium. An attempt was made to correlate changes in the albumin/alpha-2 globulin ratios and gamma globulin levels with clinical and roentgenographic status. It was observed that before treatment of the tuberculous process the albumin/alpha-2 globulin ratios were low and the gamma globulin levels were increased. As improvement occurred, albumin/alpha-2 globulin ratios increased and gamma globulin values decreased. A poor prognosis was indicated by decreasing albumin/alpha-2 globulin ratios. Electrophoretograms are of particular value in assessing the effect of surgery, especially when roentgenographic studies are not informative. They are also a valuable guide in deciding upon the duration of therapy necessary for individual cases.     

Evaluation of the prophylactic effect of gamma globulin in meningococcal infection.

Results of two controlled epidemiological tests evaluating the prophylactic effect of gamma globulin of Monogolian and Soviet production against meningococcal infection are presented. Observations were carried out on children aged 3 months to 4 years, not attending children's establishments. The results of the observation revealed the following prophylactic effect of gamma globulin of Mongolian production in the first two months after administration: index of efficiency--5.0, coefficient of efficiency--80%, P greater than 0.01. The efficiency of the prophylactic effect of Soviet gamma globulin was limited to one month: the index of efficiency amounted to 5.3, the coefficient of efficiency to 82.2%, P greater than 0.01. The course of meningococcal infection in the children who had received gamma globulin was less severe than in the children of the control group. Lethal outcome was recorded only in the group of children who had not received gamma globulin. The duration of the prophylactic effect of gamma globulin was found to depend on the height of the titres of specific antibodies in the preparation. The preparations are recommended as prophylactic means for children aged from 3 months to 4 years in doses of 1.5 ml (one dose) in the foci of meningococcal infection.     

The Metabolism of Serum Proteins in the Hen and Chick and Secretion of Serum Proteins by the Ovary of the Hen

In the chicken, serum gamma globulin (CGG) is preferentially transferred by the follicular epithelium of the ovary to the developing ova. The concentration of gamma globulin in the yolk of the unfertilized egg is many times the concentration of chicken serum albumin (CSA). This transfer occurs largely during the 4 to 5 days preceding ovulation when the growth of the ovum is most rapid. Thus, in the chicken, the follicular epithelium of the ovary serves the same purpose in the passive immunization of offspring as does the acinar epithelium of the udder in ungulates and the extraembryonic membranes in rabbits and man. The amount of gamma globulin synthesis by the chick is low during the first 2 weeks of life and is associated with low levels of serum gamma globulin. By the end of the 1st month of life, the level of serum gamma globulin increases, presumably reflecting an increased rate of synthesis. In the adult hens the half-life of I¹³¹-labeled CSA is 66 hours and that of I¹³¹-labeled CGG, 35 hours, while in the newly hatched chick for I¹³¹-labeled CSA it is 42 hours and for I¹³¹-labeled CGG, 72 hours. Thus, this species shows a gamma globulin sparing in the first days of life, as do most mammalian species.     

Protein chemical characterization of Gc globulin (vitamin D-binding protein) isoforms; Gc-1f, Gc-1s and Gc-2

Gc globulin, also called vitamin D-binding protein, is a plasma protein involved in the extracellular actin-scavenger system, vitamin D transport and possibly also other biological activities. Low levels of Gc globulin have been found to correlate with multiple organ failure and non-survival of patients with fulminant hepatic failure and trauma. Here, we characterize the dominant isoforms of plasma-derived Gc globulin from Cohn fraction IV paste with respect to amino acid sequence and posttranslational modifications. Gc globulin was purified in large scale and the isoforms separated by ion exchange chromatography. The separated isoforms and several commercial preparations of individual isoforms were characterized by mass spectrometry. This revealed that the major isoforms were non-glycosylated. Compared to the Gc-1f isoform the other dominating isoforms represented an Asp/Glu substitution (Gc-1s) and a Thr/Lys substitution (Gc-2) in agreement with DNA sequencing studies. The commercial preparations were found to represent mainly one or two isoforms. An O-linked glycan with a mass of 656 Da and terminating with a sialic acid residue was detected on a minor proportion of Gc globulin molecules.     

An electrophoretic characterization of serum proteins of the collared peccary (Tayassu tajacu)

Serum proteins of the collared peccary (Tayassu tajacu) were analyzed by agarose gel electrophoresis for wild adult males and females, nursing young, and reproductively-active females in captivity. Electrophoretic profiles of the adult peccary showed at least six distinct protein bands corresponding to the fractions: albumin, alpha-1, alpha-2, beta-1, beta-2, and gamma globulin. Globulin fractions of the peccary had different mobilities from the domestic swine. The only sexual dimorphisms were associated with the beta globulin:albumin ratio and the albumin:globulin ratio. Ingestion of colostrum in 1-day-old neonates was marked by a very large increase in gamma globulins. The only significant difference between pregnant and lactating females was in the alpha globulin:beta globulin ratio. Lactating females had higher concentrations of alpha-2 globulin than non-pregnant females.     

Advisability of carrying out mass prevention of infectious hepatitis with gamma globulin

In Vilnius 15,183 pupils in 20 schools were observed for two years; in October, 1966, gamma globulin prophylaxis of infectious hepatitis was carried out among all the pupils of these schools. 17.062 pupils in 27 schools, who received no gamma globulin, were used for control. At the end of the first year of observation morbidity rate among the immunized children was found to be considerably lower than among the school children of the control group. During the second year of observation morbidity rate among the children of the "old" immunized group increased 3.5-fold and did not significantly differ from morbidity rate among the children of the control group. The latter increased 1.8-fold. The authors have come to the conclusion that the effect of gamma globulin prophylaxis on the epidemic process depends on the method of such prophylaxis. The method of prophylaxis by introducing gamma globulin immunization with universal coverage, as the use of the former method is not followed by an increase in total morbidity (i.e. among both immunized and nonimmunized children) during the second year after immunization, which is observed after prophylaxis by universal immunization.     

Natural antibodies against Pseudomonas aeruginosa in human gamma globulin preparations

A total of 191 batches of commercial human gamma globulin were studied with the aim of detecting natural antibodies by means of cross and countercurrent electrophoresis, immunodiffusion in agar gel and the passive hemagglutination test. Commercial human gamma globulin preparations were found to contain, to a greater or lesser extent, natural antibodies to the cell-wall O-antigen of Ps. aeruginosa and the antigens of its extracellular slime (capsule); the titers of these antibodies were 1 : 64 to 1 : 256 in 75-85% of cases, 1 : 512 and greater in 10-11% of cases. Commercial gamma globulin preparations also contained antibodies protecting mice from experimental Pseudomonas infection. No correlation was detected between the degree of serological activity and the protective activity of gamma globulin preparations.     

Aphelenchoides rutgersi: Growth requirements for heme and purine nucleotides in crude chick embryo extract

The crude chick embryo extract (CEE) at 20% concentration (v/v) in the basal medium (M-10), enabled the growth, reproduction, and continuous culturing of Aphelenchoides rutgersi Hooper and Myers. No such growth promoting effect was found with chick embryo extract (Ultra filtrate), human gamma globulin and beta lipoprotein (Cohn fraction IV-4) when used either alone or in combination with hemin.The nematode growth factor(s) in the CEE were stable to trypsinization and temperature of 100 C for 5 min but not for 15 min. After acid treatment to pH 3.0 with sulfuric acid, the supernatant retained only partial activity. But, after treatment with perchloric acid to pH 3.0 or with activated charcoal, the CEE was completely ineffective as a growth supplement. The activity of charcoal-treated CEE has been completely recovered by addition of hemin (10 μg/ml M-10).Deletion of nucleic acid supplements from M-10 but not from the CEE did not affect the reproduction of the nematodes at all. However, the reproduction was significantly reduced when the nucleic acids were removed both from the M-10 and the CEE. Selective deletion of deoxyribonucleotides and purine-, and pyrimidinenucleotides of ribose series indicated only limited nutritional requirement for purine compounds.     

Development of resistance in rabbits to immature stages of the Ixodid tick Rhipicephalus appendiculatus

A study of acquired resistance in rabbits to larvae and nymphs of Rhipicephalus appendiculatus Neumann showed a positive correlation between the levels of gamma globulin in the serum and the resistance developed by the host. A negative correlation between the weight of engorged ticks and gamma globulin levels was also demonstrated during this study. Repeated infestations with ticks evoked a typical antibody response to antigenic challenge. The levels of gamma globulin stabilized after three infestations and no further decrease in the weight of ticks was observed. The mechanism of the acquisition of resistance by the rabbits, the production of antibodies and the involvement of complement are discussed.     

Development and trial of a heterogenous antistaphylococcal gamma-globulin

Antitoxic and antibacterial equine gamma globulin for the treatment of staphylococcal infection has been developed and introduced into medical practice. The pronounced effectiveness of the preparation has been demonstrated in comparison with the ineffectiveness of traditional therapeutic measures. The development of mild forms of the serum disease in 33% of cases indicates that further investigations on the purification of gamma globulin preparations are necessary.     

Human alpha-chain globin messenger: prediction of a nucleotide sequence

The presence in human serum of inhibitory activity to rat liver insulin specific protease has been detected in an alpha₁ globulin preparation (Cohn Fraction IV₁). Separation into four components and partial purification (40 to 107 fold) has been achieved by heat denaturation of non-active protein, Sephadex G-100 gel filtration and ion-exchange chromatography upon QAE Sephadex. Each of the inhibitors was found to be competitive in nature. The molecular weight of the inhibitors is between 4,000–7,000 and the activity is destroyed for the most part by chymotrypsin.     

Effectiveness of the seroprophylaxis of tick-borne encephalitis depending on the titer of homologous gamma-globulin antihemagglutinins

To give a rationale for using homologous gamma globulin with antihemagglutinin titers of 1 : 20 to 1 : 80 for the prophylaxis of tick-borne encephalitis, 5-year observations covering all persons attacked by ticks in one of the intensive natural foci of the disease in the Western Urals have been made. The threefold statistically significant difference in the morbidity rate of tick-borne encephalitis between groups of persons immunized and not immunized with gamma globulin has been shown.     

Effect of anti-sperm gamma globulins on metabolism of human spermatozoa.

Rabbits were immunized with saline extracts of human spermatozoa Presence of antibodies to spermatozoa was confirmed by Ouchterlony gel diffusion, microscopic sperm agglutination, and immunofluorescent techniques. Anti-sperm gamma globulin significantly decreased the average oxygen consumption of 10⁸ washed human spermatozoa. Sperm incubated in the presence of anti-sperm gamma globulin had a significantly lower rate of lactic acid accumulation than sperm incubated in normal rabbit gamma globulin. It appears that anti-sperm antibodies can influence both aerobic respiration and glycolysis of washed human spermatozoa.