[Lectin-enzyme assay as a method of estimation of immunoglobulins' glycosylation].

The mechanism of interaction of lectins with IgG molecules by the method of the lectin-enzyme assay has been described that allows to register a degree of human serum IgG molecules' glycosylation (mannosylation in case of lectin of Pisum sativum) in norm and at pathology. To detect an authentic difference in a glycosylation degree between control and pathological IgG, the wells of an ELISA plate were coated with an antibody in concentration of 1 microg/ml. Introducing alpha-D-mannose between the stages of incubation of immunoglobulin and lectin showed, that alpha-D-mannose inhibits the affinity of lectins for IgG. The preliminary incubation of lectin with IgG molecules stabilizes the activity of horseradish peroxidase, which labeled the lectins. Lectin-enzyme assay, in which Fab and Fc fragments of IgG were used, showed that lectin of Pisum sativum possesses a higher affinity for Fab regions. These findings and the glycosylation analysis of paraproteins and Bence-Jones proteins of multiple myeloma patients help to understand the details of interaction of immunoglobulins and lectins.

Infusion of Fc gamma fragments for treatment of children with acute immune thrombocytopenic purpura.

Treatment of acute immune thrombocytopenic purpura (ITP) with intravenous immunoglobulin (IVIG) induces partial or complete responses, shown by transient or persistent increases in platelet count. The clinical benefit could be due to blockade of the Fc gamma receptor (Fc gamma R); platelets sensitised by IgG could not be cleared by cells of the reticuloendothelial system if Fc gamma R on these cells was blocked with IVIG. To find out whether this putative mechanism is correct, we treated twelve children who had acute ITP with intravenous infusions of Fc gamma fragments. Eleven children showed rapid increases in platelet counts to above the critical value of 50 x 10(9)/L, thereby avoiding major haemorrhagic risk. The response was stable in six patients and transient in five. No adverse reactions were observed. In responders who had detectable platelet-associated IgG before treatment (> 1500 IgG per platelet), platelet IgG fell substantially with treatment. Serum soluble CD16 (sCD16 or sFc gamma RIII) concentrations, measured in five children, showed transient or stable increases that correlated with the rise in platelet count. No sCD16 was detected in the Fc gamma preparation used. We conclude that the infusion of Fc gamma fragments is an efficient treatment of acute ITP in children. The efficacy of Fc gamma fragments strengthens the hypothesis that Fc gamma R blockade is the main mechanism of action of IVIG in ITP, although other immunoregulatory mechanisms triggered by the presence of increased sCD16 concentrations in serum could be involved in the clinical benefit observed.

Urinary excretion of immunoglobulins and their subunits in human subjects before and after exercise.

Immunoglobulins and their subunits in urines collected before and after exercise from healthy human subjects were studied. Quantitative analysis showed that only gamma A- and gamma G-immunoglobulins were excreted as whole molecules in normal urine, at a concentration of 0.35 and 1.70 micrograms/min., respectively. Exercise enhanced the excretion of both types of molecules. In some cases, gamma D-immunoglobulins were found in "exercise urine." Gel filtration on Sephadex G-150 showed that only gamma G-subunits were present in normal and in "exercise urine." The distribution of gamma G-subunits was not affected by exercise. The authors consider the importance of the present data in relation to renal physiology.

A comparison of iodine and indium labelled anti CEA intact antibody, F(ab)2 and Fab fragments by imaging tumour xenografts.

An intact anti CEA monoclonal antibody (C198) and its F(ab)2 and Fab fragments have been radiolabelled with 131I and 111In and their biodistributions and tumour imaging capabilities examined in mice with human tumour xenografts. 131I labelled F(ab)2 and Fab fragments showed improved tumour visualization compared with intact antibody, principally because of the more rapid whole body elimination of the radiolabel. Studies using 111In labelled fragments demonstrated that a major proportion (greater than 60%) of the administered radioactivity was retained in the kidneys and this was detrimental to tumour imaging in the mouse xenograft model. The present study emphasizes the importance of selecting the most appropriate combination of antibody preparation and radiolabel, and that the choice of radionuclide is influenced by both its physical and subsequent pharmacological characteristics.

Generalized Castleman's disease with urinary elimination of heavy chain fragments.

A generalized form of Castleman's disease of transitional type is reported in a 66-year-old male who was found in addition to have gamma heavy chain fragments in his urine. One portion of them was constituted by Fc fragments and the other ones by fragments which reacted with anti-Fab/IgG and anti-Fc/IgG antisera but not with anti-kappa and anti-lambda antisera. The findings of the present case support an immunological basis for Castleman's disease.

Identification and incidence of a urinary fragment of IgG.

Urine from some patients, when concentrated approximately three hundred fold and immunoelectrophoresed against anti-IgG, shows an unexpected additional precipitin line in the alpha-globulin region. This reaction has been shown to be due to the presence of a low molecular weight (approximately 20 000) fragment of the heavy chain of IgG. A retrospective examination of immunoelectrophoretic plates run over a period of five years has revealed that this fragment was present in 30 out of 110 patients.

Catabolism and biologic properties of two species of rat IgG2a Fc fragments.

Prolonged papain digestion of rat IgG2a has recently been shown to yield two species of Fc fragments, termed Fc(I) AND Fc(II). The results of structural studies indicated that Fc(II) fragment was 15 to 20% smaller than Fc(I), probably secondary to loss of a carboxy-terminal peptide. The effects of these structural laterations on the catabolism and biologic properties of the Fc fragments were determined. The results of catabolic experiments indicated that after injection into normal rats Fc(I) fragments were retained in the circulation and slowly catabolized whereas Fc(II) fragments rapidly underwent filtration in the kidneys. In nephrectomized rats, however, both Fc(I) and Fc(II) fragments possessed identical slow rates of catabolism, as determined by serum disappearance and whole body catabolic experiments. Fragment pFc, corresponding to Cgamma3 domain, was different from either of the Fc fragments in exhibiting rapid rates of catabolism in both normal and nephrectomized rats. Fc(I) was more active in complement fixation and in adherence to the Fc receptor on monocytes in comparison with Fc(II). These results support the conclusion that catabolism of Fc and maintenance in circulation are separate processes influenced by different structures in the Fc fragment. The catabolism of Fc is controlled by structures in the Cgamma2 domain. This process probably is not related either to complement fixation or to adherence to the Fc receptor on monocytes. Some correlations between the structure and biologic properties of these Fc fragments are discussed.

Serum disappearance and catabolism of homologous immunoglobulin fragments in rats.

The serum disappearance, metabolic clearance and whole body catabolism of homologous immunoglobulin fragments were studied in rats. The rapid disappearance of Fab fragments from serum in normal animals was no longer present after nephrectomy. In contrast, the serum disappearance curve of Fc fragments was not altered by nephrectomy. The results of three different experiments, however, indicated that similar to Fab fragments, some Fc fragments underwent filtration and degradation in the kidneys. First, the amount of intact Fc fragments excreted in the first day after injection increased from 6% of the injected dose in normal rats to 17% in rats pretreated with sodium maleate. Secondly, nephrectomy eliminated the rapid phase of whole body catabolism of inected Fc fragments. Thirdly, auto-radiographic studies showed localization of Fc fragments in the renal proximal tubule cells in the first 3 hr after injection. An identical localization was seen with Fab fragments. These results support the conclusion that removal from circulation by glomerular filtration and subsequent reabsorption and degradation in proximal tubule cells represent the major mechanism for catabolism of Fab fragments. Although some Fc fragments undergo the same fate, most injected Fc fragments equilibrate with unknown sites where they are possibly bound and made unavailable for filtration in the kidneys.