Comparison of IgG preparations by a turbidimetric assay of opsonizing capacity.

A convenient turbidimetric phagocytosis assay was applied for the functional comparison of various intravenous IgG preparations. Staphylococcus aureus (Oxford) was opsonized by the immunoglobulin samples in the presence of an IgG deficient serum as a source of complement. The opsonized bacteria were subjected to phagocytosis by neutrophil granulocytes isolated from healthy adults. The time course of phagocytosis was monitored by the decrease of light absorbance at 400 nm. Changes in light absorbance during a 15 min period of opsonophagocytosis (delta E(400)) were expressed as a percentage of delta E(400) obtained by a reference IgG preparation. The opsonizing effect of five commercially available i.v. IgG preparations was compared. Three different preparations containing whole, non-modified IgG molecules had a comparable opsonizing effect while a further one prepared by propiolacton modification displayed a reduced activity (52%) of the reference preparation, taken as 100%. A preparation consisting of IgG molecules without an Fc-region proved to be practically ineffective (8.7%).

Comparison of hypotensive response to aggregated IgG or to bacterial LPS in rats.

OBJECTIVE AND DESIGN: Rats treated with aggregated IgG (Aggr.) become "refractory" to the hypotensive action of a second dose of Aggr. The objective of this study was to assess the responsiveness of animals pretreated with Aggr. to bacterial LPS and vice versa. MATERIAL OR SUBJECTS: Female Wistar rats (250-300 g) were used. Each experiment was carried on at least 4 animals. TREATMENT: A human IgG preparation containing 30% aggregates (10-16 mg/100 g) or E. coli serotype 0111.B4 (0.005-mg/100 g) was administered i.v. Certain groups of animals were pretreated with 1 mg/100 g GdCl3 or with 10 mg/100 g pentoxyphylline (PTX). METHODS: Arterial blood pressure was monitored in the carotis-using a polyethylene cannula and an electronic tension meter. Tumor necrosis factor alpha (TNF-alpha) activity was estimated by the use of an L-929 cell cytotoxicity assay. RESULTS: Pretreatment of rats with a sublethal dose of LPS impaired the hypotensive reaction of the animals to Aggr. Rats male "refractory" to Aggr. reacted to the injection of LPS with hypotension and a second phase milder than in the controls. Hypotension could not be elicited by Aggr. in rats pretreated with GdCl3. The same pretreatment had no effect on the first phase of hypotension induced by intravenous injection of LPS, whilst a mitigation of the second phase was observed. Infusion of PTX immediately prior to Aggr. administration prevented the drop of blood pressure. A sizeable level of TNF-alpha was detected only later than blood pressure had reached its minimum level following Aggr. administration. CONCLUSIONS: Hypotension induced by LPS may involve a macrophage population broader than that responsible for the vascular action of Aggr. The data presented do not support a primary role for TNF-alpha in Aggr. induced hypotension.

Sodium-cromoglycate (Cromolyn) selectively increases the binding and phagocytosis of unsensitized target cells by rat peritoneal macrophages.

The influence of sodium-cromoglycate (cromolyn) on the binding and ingestion of sheep erythrocytes (SRBC) by elicited rat peritoneal macrophages (M phi) was studied using unsensitized SRBC. SRBC sensitized by homologous IgG or by IgM and complement as target cells. Preincubation of M phi with the drug (1 nM/1-2 mM/1) markedly enhanced both binding and ingestion of uncoated SRBC. The IgG-related increment in binding and phagocytosis was not significantly influenced by the drug. When target cells were coated by IgM and complement cromolyn pretreatment was ineffective. Preincubation of M phi by bovine brain gangliosides (BBG) diminished the cromolyn-induced enhancement of target cell binding and phagocytosis. When SRBC were pretreated by BBG, an increase of binding and phagocytosis was observed. These data suggest that cromoglycate may enhance the capacity of M phi to bind erythrocytes via ganglioside structures. Coating SRBC by complement components appears to interfere with binding of erythrocytes to M phi ganglioside receptors.

Survey of major genotypes and subtypes of hepatitis C virus using RFLP of sequences amplified from the 5' non-coding region.

A method is described for identifying different genotypes of hepatitis C virus (HCV) by restriction endonuclease cleavage of sequences amplified by PCR from the 5' non-coding region. Using the enzymes HaeIII-RsaI and HinfI-MvaI, followed by cleavage with BstU1 or ScrFI, it was possible to identify and distinguish HCV genotypes 1a, 1b, 2a, 2b, 3a, 3b, 4, 5 and 6. The method was used to investigate the prevalence of these genotypes in 723 blood donors in 15 countries, the largest survey to date, and one which covered a wide range of geographical regions (Europe, America, Africa and Asia). These results, combined with a review of the existing literature, indicate the existence of several distinct regional patterns of HCV genotype distribution, and provide the framework for future detailed epidemiological investigations of HCV transmission.

HCV antibodies in Hungarian blood donations. Experiences collected by ELISA tests, immunoblot assays and polymerase chain reaction and protocols for donor management.

Routine screening of Hungarian blood donors for anti-HCV commenced in the second half of 1992. Before this, five available anti-HCV ELISA kits were compared in pilot studies. In the first series, 831 random donor samples were tested by one of the tests and the 12 (1.4%) reactives found were retested by the other four. Six of the reactives were positive in all ELISA. In the second series, 325 samples from donors with elevated transaminase levels were tested by all five kits. Forty-four were found to be reactive by one or more of the tests and 32 (10%) were positive in all five assays. Samples concordantly reactive in the ELISA were positive in second or third generation recombinant immunoblot assay (RIBA 2 or RIBA 3); those that gave discordant results were indeterminate or negative. Eleven concordantly reactive samples from the second series were HCV RNA positive by polymerase chain reaction (PCR). In the first period of screening with Abbott ELISA 2 a repeat-reactivity rate of 0.98% was observed in 171,106 samples tested. Reactives were retested for supplementary testing by Wellcozyme anti-HCV. Donors reactive in both tests and strongly reactive (ELISA ratio (ER) = optical density/cut off > or = 2.5) in either of them were permanently deferred. Those negative in the supplementary ELISA or weakly reactive (1.0 < or = ER < 2.5) in both tests were subjected to RIBA 2. On the basis of RIBA, positive donors were permanently deferred, indeterminates were excluded for 1 year and negatives were readmitted. In 1992, 1,347 supplementary tests were completed; 824 (61%) of the respective donors were permanently deferred, 218 (16%) were deferred for 1 year and 305 (23%) were readmitted.

Hypotensive action of aggregated IgG in rats. Study of the unresponsiveness to a second dose following restoration of the blood pressure.

Human IgG preparations containing aggregates have been reported to induce hypotension in rats. Animals surviving a hypotensive dose of such a preparation were found unresponsive to a second dose given the next day. Unresponsiveness was induced by an isolated fraction of aggregated IgG, but not by purified monomers. Preparations containing aggregated and monomeric IgG appeared more efficient in inducing unresponsiveness than aggregates alone. The phagocyte function in vivo was assessed in unresponsive animals. No significant difference was found in the carbon clearance and in the human erythrocyte clearance test suggesting that a general impairment of the phagocyte function is not necessary for unresponsiveness. Previous studies indicated the involvement of PAF in hypotension by aggregated IgG. Hypotension was elicited by the injection of PAF in rats made "refractory" to aggregated IgG like in controls.

Rat IgG subclasses mediating binding and phagocytosis of target cells by homologous macrophages.

Attachment and ingestion of 51Cr-labelled TNP-SRBC sensitized by rat IgG1, IgG2a or IgG2b-type antibodies by homologous, elicited peritoneal macrophages were studied. IgG1 was found to be the most efficient isotype in mediating these functions. The antibody doses required for a significant attachment were found to differ with the isotype of Ab, while doses needed for a significant phagocytosis and antibody-dependent cellular cytotoxicity (ADCC) varied between 400-700 Ab/SRBC with all the isotypes studied. Both binding and phagocytosis were also influenced by the degree of hapten conjugation when target cells were sensitized by IgG1. Inhibition of these functions by soluble immune complexes and monomeric immunoglobulins suggests the involvement of two Fc gamma R in binding of the three isotypes. Based on the present work and on previous results we conclude that IgG2a interacts with a receptor binding complexed IgG only (Fc gamma RII), IgG2b binds to a different receptor which appears to bind monomeric ligand as well (Fc gamma RI), while IgG1 seems to interact with both types of receptor. We propose that phagocytosis can be mediated by both Fc gamma RI and Fc gamma RII.

[Reactivity of hepatitis C virus (HCV) antibody among blood donors in Hungary]. / Hepatitis C vírus (HCV) ellenanyag reaktivitás a hazai véradók körében.

The anti-HCV results of 1958 donors are discussed. The Elisa positivity of high risk subjects was as follows: 60% in coagulopathies, 14% in transfused haematological patients and 32% in patients on dialysis. Elisa reactivity of random blood donors was 1.82%, in the group, selected on the basis of transaminase positivity it was over 8%. The confirmed results were compared by the commercially available Elisa tests. 30 positive samples were confirmed by RIBA or Abbott confirmation assay. 35 of the 55 anti-HCV positives were reactive by all the Elisa, 19 samples could be confirmed, 25 were not subjected to any confirmatory assay. Further studies, and the introduction of mandatory screening of blood donors for anti-HCV are necessary.

[The role of HCV in the pathogenesis of post-transfusion hepatitis]. / A HCV szerepe a poszttranszfúziós hepatitisek kórokában.

Between January 1987 and April 1988 following 462 open heart surgery operations, 83 patients with icteric hepatitis were seen. Among them 59 patients were anti-HCV positive with the Ortho anti-HCV test, these findings suggested a hepatitis C virus infection. The source of viral infection was searched, and a prothrombin complex concentrate which had been used during that period, seemed to be the potential cause of bloodborne hepatitis C. The results suggest that special care is required when using such blood products.

Antibody mediated lysis of hapten-conjugated target cells by macrophages and by complement: the influence of IgG subclass, antibody and hapten density.

Lysis of 51Cr-labeled TNP-SRBC sensitised by rat IgG1 or IgG2a type antibodies by homologous, paraffin oil-elicited peritoneal macrophages (ADCC) or by homologous complement was studied. IgG2a was found to be markedly more efficient in mediating both ADCC and complement dependent lysis compared to IgG1. Inhibition of the ADCC pointed to the involvement of separate but partially overlapping interaction sites for the two isotypes. We suggest that FcRII type receptors play a favoured role in both IgG2a and IgG1 mediated ADCC. The threshold amount of bound antibody required for ADCC was lower than that sufficient for complement dependent lysis regardless on the subclass or on hapten density. The extent of lysis (both ways) was found to depend on hapten density using equal amounts of antibody. The results are interpreted as terms of the possible requirements for association of IgG molecules on the target cell surface.

Interaction between rat peritoneal macrophages and sensitised erythrocytes: dependence on IgG subclass, antibody density and the degree of hapten conjugation.

The interaction between macrophages (M phi) and antibody sensitised target cells was studied by the use of rat peritoneal macrophages, TNP hapten conjugated sheep red blood cells (SRBC) and homologous antibodies of subclasses IgG1 and IgG2a. Under optimal conditions, the great majority of the M phi formed rosettes with IgG1-sensitised antibodies while a maximum of 50% was achieved when target cells were sensitised by IgG2a. Using a double rosette technique, the major part of rosette-forming cells was found to bind both of the isotypes. IgG1-mediated rosette formation was observed at very low degrees of sensitisation as opposed to IgG2a-mediated target cell binding. Not only the amount of bound antibody but also the degree of hapten conjugation (epitope density) appear to influence the ratio of rosette-forming cells. IgG1-mediated rosette formation was partially inhibited by monomeric IgG1 and more efficiently by soluble ovalbumin (OVA)-anti-OVA complexes involving IgG1-type antibodies, while IgG2a mediated rosette formation was inhibited by OVA-anti-OVA complexes containing IgG2a type antibodies, and less efficiently by complexes involving IgG1. No inhibition was found by monomeric IgG2a. Based on the present data, we propose that two types of receptors are involved in the interaction of M phi and target cells coated by IgG1 and/or IgG2a type antibodies. One requires a multiple antibody-receptor interaction, binding both subclasses at overlapping binding sites; the other is able to interact with IgG1 and does not depend on the multiplicity of interactions.

[Hepatitis C virus antibody in Hungarian blood donors, persons at risk and patients with post-transfusion hepatitis]. / Hepatitis C vírus ellenanyag hazai véradókban, veszélyeztetettekbenés poszt-transzfúziós hepatitisben megbetegedettekben.

The authors screened blood donors, persons at high risk of infection and patients with post-transfusion hepatitis by the use of the new hepatitis C antibody test. 1.7% of persons donating blood acceptable for use according to current criteria were found positive. This ratio was markedly higher among donors displaying an elevated transaminase level. Among the various patients tested 85% of the haemophiliacs, 14% of the transfused haematology patients, and 40% of those on dialysis were found positive. In two groups of patients with post-transfusion non-A, non-B hepatitis the ratio of positives was 69% and 92%, respectively. The frequency of hepatitis C antibody was not sizably higher in the health personnel tested than that found in blood donors. The results indicate hepatitis C virus to be the main source of post-transfusion non-A, non-B hepatitis in Hungary too. As expected, HBc antibody revealing a previous contact with hepatitis B virus was found very frequent in patients with coagulopathies and in those on dialysis. A relatively high anti-HBc prevalence was found among health personnel and blood donors too. Authors intend to emphasize the need of thorough consideration of indications in hemotherapy although improvements in the screening of donations can diminish the risk of virus transmission by blood or blood derivatives.

Hypotensive action of IgG preparations containing aggregates is suppressed by PAF-receptor antagonist BN 52021 and by gadolinium chloride (an agent blocking Kupffer cell function).

The hypotensive side effect of four IgG preparations was studied in a rat model. An unmodified, chromatographically isolated preparation (prepn. C) appeared to induce a transient drop in blood pressure via the kininogen kinin system. A preparation made by ethanol fractionation (prepn. E) and an ethanol-fractionated, modified product (prepn. M) produced an action which does not depend on kininogen activation. No blocking of kininase was required for this effect and it was brought about by prepn. M was devoid of any sizeable prekallikrein activator or kallikrein like proteinase content. The effect was associated by the aggregate fraction of prepn. E, but not with IgG monomers of the same preparation. Animals given these latter types of IgG preparations were refractory to a second dose 24 hours later. The involvement of PAF in the kinin-independent hypotensive action is suggested by the finding that PAF-receptor antagonist BN 52021 prevented the effect and returned the blood pressure close to normality when given during hypotension. A blockade of Kupffer cells brought about by administration of GdCl3 was also found to prevent the effect pointing to the involvement of these type of cells in the reactions.

Fine specificity of a rabbit antibody interacting with human IgG Fc receptor-like molecules.

A polyclonal rabbit antibody raised against an Fc receptor (FcR)-like membrane glycoprotein fraction of chronic leukaemic lymphocytes has previously been prepared and partially characterized. This antibody, called AbA, was found to precipitate a 70-kDa and a 45-kDa fraction of the detergent lysate of U937 cells and to inhibit ligand binding to Fc gamma R on the P388D1 murine macrophage cell line. In the present work we have characterised this antibody further. All Fc gamma RII-positive B lymphoblastoid cell lines, as well as resting human B lymphocytes, were positively stained with the AbA antibody. U937 cells were found to be negative, but after stimulation with phorbol ester (PMA), 50% of the cells became positive. AbA antibody did not react with human T cell lines or with the T + 0 cell subset of peripheral blood. Monocytes were also negative. On the other hand, AbA antibody exhibited a dose-dependent inhibition of antibody-mediated cytotoxic reaction (ADCC) of monocytes, while not affecting K cell-mediated ADCC. It had an inhibitory effect of EA rosette formation of B cells and stimulated U937 cells. Furthermore, it interacted with the soluble form of Fc gamma RII released by activated B lymphocytes, and--similarly to IgG--precipitated a 33 kDa fraction from the supernatant of B cells.

IgE levels in normal human sera and IgG preparations.

Serum IgE levels were determined in 318 Hungarian blood donors by PRIST technique. Age-dependent decrease of IgE and higher values in males than in females were found. Commercial IgG preparations contained variable amounts of IgE related to the procedures for purification. The normal IgE level in healthy Hungarian adults were compared to the reported normal values of other countries.

Safety aspects of intravenous immunoglobulins.

Seven commercially available intravenous immunoglobulins (IVIG) preparations, a gamma globulin prepared by ethanol fractionation, and an experimental IgG isolated by a chromatographic procedure were compared in several tests. Split products were present in preparations manufactured by procedures involving protease treatment and in a sulphitolysed IgG. The same preparations and another chemically modified product displayed a loss in their capacity to bind staphylococcal protein A. None of the preparations exerted a high anticomplementary activity using concentrated human serum as a complement source. No strict correlation between aggregate content and anticomplementary activity could be established. None of the commercial IVIG preparations tested displayed a sizeable hypotensive action as assessed by a rat model involving potentiation of bradykinin action by an angiotensin convertase inhibitor. The chromatographically purified IgG and an intramuscular IgG prepared by Rivanol fractionation contained high endogenous protease and prekallikrein activator (PKA) activity, respectively and both were found markedly hypotensive. Neither endogenous protease nor PKA activity was detected in the Cohngammaglobulin fraction. However, it was very strongly hypotensive even without any previous blocking of angiotensin convertase. Our data support the view that immunoglobulin preparations may affect blood pressure without inducing bradykinin generation.

Prevalence of HIV-antibodies in patients with haemophilia in Hungary.

Incidence of HIV antibodies have been studied in 617 patients with congenital bleeding disorders. Screening was performed with the Organon ELISA test, repeatedly positive samples were tested with four different confirmatory assays. HIV antibodies were found in 3/356 patients with haemophilia A, 21/114 patients with haemophilia B, 3/123 patients with von Willebrand disease and in 1/24 patients with other types of congenital coagulation disorders. All the 28 seropositive patients were treated with imported coagulation factor, mostly with factor IX concentrates. No patient treated exclusively with nationally produced factor preparations between 1981 and 1985 was found to be anti-HIV positive.