M. Kávai

Factor XIII of blood coagulation in human monocytes

The presence of Factor XIII subunit a was demonstrated in human monocytes by immunoperoxidase staining using specific antisera against Factor XIII and its subunits. This finding was verified by immunobiochemical techniques, as well. In an immunoblotting system after SDS polyacrylamide gel electrophoresis of denatured monocyte homogenate a protein band comigrating with Factor XIII subunit a showed positive reaction with antibodies against this subunit or whole Factor XIII. In contrast, no subunit b of Factor XIII could be detected by either of these methods in monocytes. Activity measurements were carried out by the dansylcadaverine incorporation assay in the absence and presence of anti-Factor XIII antibody with and without thrombin activation. The expression of transglutaminase activity required thrombin and was completely abolished in presence of anti- Factor XIII antibody, which clearly indicate that practically all the transglutaminase activity measured in monocytes comes from Factor XIII. Factor XIII of monocytes and macrophages might have a role in formation of focal fibrin thrombi as well as in organization of stable, fibrinolysis resistant fibrin clot at the site of inflammation or around tumor cells.

Enzyme-linked immunosorbent assay for antibodies to native DNA in sera of patients with SLE

A micro-ELISA technique has been developed to measure antibodies to native DNA and used in SLE patients. The distribution of antibody to native DNA in the main immunoglobulin classes was studied, using anti-human globulin conjugates labelled with peroxidase. the antigen (double-stranded DNA from calf thymus) used in the assay was adsorbed to the surface of polystyrene plates treated with methylated bovine serum albumin. The standardization of the method was carried out by use of globulin calibration curves.

Circulating immune complexes and monocyte Fc function in autoimmune diseases.

The phagocytosis of separated and adherent monocytes of patients with systemic lupus erythematosus is subnormal as compared to controls on the basis of latex and yeast uptake. The monocytes from the same patients react with antibody-coated sheep red blood cells in a significantly higher degree than normal monocytes. There is a correlation between the percentage of reactive monocytes and the serum immune complex content. After brief treatment of patients with levamisole the phagocytic function of monocytes is restored and at the same time the circulating immune complex content is decreased.

Immune complex clearance by complement receptor type 1 in SLE

Patients with systemic lupus erythematosus (SLE) have relative deficiencies of the C3b/C4b receptor (CR1, CD35) on erythrocytes (E). This receptor takes part in the binding, transport and endocytosis of circulating immune complex bound complement components (ICC). Besides the autoantibodies the abnormalities in IC elimination are fundamental to the pathogenesis of SLE. During the last 15 years more than 100 patients with SLE have been treated in our Department and their data on ICC clearance by ECR1 analyzed. After plasmapheresis the ECR1 expression and also the binding sites for ICC were increased, while the level of IC and autoantibodies were reduced. Stimulating erythropoiesis in patients with anaemia and lupus nephritis caused the decreased expression and functional activity of ECR1 to be improved. In patients with SLE the level of soluble CR1 was also decreased, but a significant portion of soluble CR1 bound ICC in vivo, especially in those with severe renal lesion.

Immunological Investigations in Acute and Chronic Human Pancreatitis

Follow-up immunological studies in 27 patients with acute pancreatitis of known etiology showed a significant elevation in the level of circulating immune complexes (IC), a significant inhibition in migration of leukocytes (with direct LMT) of patients and a significant decrease in the percentage of T-active, T-total peripheral lymphocytes and in the absolute count of peripheral T cells. Elevated circulating IC levels could been detected 3-4 weeks after the onset of acute pancreatitis. These immunological changes have still been demonstrated in a number of patients 7-14 months after recovery. We have found similar immunological alterations in patients with chronic pancreatitis as well. The possible causes and role of these long-term existing immunologic abnormalities are discussed.

CR1 density polymorphism and expression on erythrocytes of patients with systemic lupus erythematosus.

The present study investigated the expressed number of CR1 on erythrocytes (E) in relationship of the CR1 density genotype from 46 patients with systemic lupus erythematosus (SLE) and 47 healthy volunteers. The CR1 genotype was determined by a method based on polymerase chain reaction (PCR) amplification of the genomic DNA fragment of 1.8 kb separated by HindIII endonuclease digestion and agarose gel electrophoresis. Our data supported the earlier results that the number of binding sites/E for monoclonal anti-CR1 decreased among SLE patients compared with normal individuals having the same alleles for the CR1/E density. At the same time the novelty of our recent results was that the decreased expression of CR1 on E correlated significantly with kidney involvement in patients homozygous for the CR1/E high density allele (HH). These data suggest that the deficiency of the detectable number of CR1 on erythrocytes is acquired in this SLE population.

Triggering of respiratory burst by phagocytosis in monocytes of patients with systemic lupus erythematosus.

The triggering of the respiratory burst by phagocytosis via different receptors in monocytes of patients with systemic lupus erythematosus (SLE) was investigated. The superoxide anion synthesis was assayed by reduction of fcrricytochrome C that was inhibited by superoxide dismutase. The mononuclear cell suspensions were triggered by IgG‐coated latex. C3 complement fragment‐coated and uncoated yeast (Saccharomyces cerevisiae). Superoxide generation induced by phagocytosis via FcγR was decreased in monocytes of patients with SLE. On the other hand. MoAbs against FcγRl, FcγRll and especially CR3 could also induce superoxide anion synthesis. At the same time, superoxide generation induced by anti‐CR3 could be inhibited with C3‐coated yeast.

Evaluation of different methods for detecting circulating immune complexes. Studies in patients with lung cancer

In a collaborative study involving 7 laboratories, sera from 53 patients with lung cancer, 37 primary and 16 secondary tumours, and sera of 40 healthy blood donors were tested by 19 different assays or assay modifications used for detecting immune complexes. In 12 out of 19 assays, significantly higher immune complex levels were found in the cancer patients than in the healthy subjects. Assays based on interactions between immune complexes and Fc receptors of different cells (lymphocytes, macrophages of platelets) discriminated between cancer patients and health subjects and a high percentage (47-87%) of positivity was observed in such assays in patients with lung cancer. In contrast, none of the tests based on immune complex-complement interactions discriminated between cancer patients and health subjects. Immunochemical analyses of the PEG precipitates obtained from the sera tested revealed that the concentrations of IgG, IgA and C3 were significantly higher in the precipitates obtained from patients sera than from control sera, but no significant differences were seen in IgM and C1q concentrations. A 100% correct classification of individuals tested was obtained on discriminant analysis of results with 3 assays: EA rosette inhibition, ADCC inhibition and C3 concentration in PEG precipitates. Correlation between results obtained with individual sera by the different assays was very poor: significant correlation coefficients were found in only 13% of all possible paired comparisons. Our results suggest that Fc receptor-dependent assays are more suitable for detection and measurement of circulating immune complexes in lung cancer than tests based on interactions with complement.

Chemotactic and stimulating effect of tuftsin and its analogues on human monocytes

Abstract Tuftsin, known for its phagocytosis stimulating capacity was examined for its chemotactic activity using a MN cell migration assay based on the Boyden technique. Tuftsin functioned as a chemotactic agent for MN cells in a dose-dependent way with highest activity detected in the concentration range of 50–200 μg/ml tetrapeptide. Pre-incubation of monocytes with tuftsin enhanced locomotion in tests of both random migration and leucotaxis. Structure-activity studies were carried out with [Ala 1 ]-, [Ser 1 ]-, [D-Ser 1 ]-, [Phe 3 ]-, [Phe 4 ]-, [D-Ala 4 ]- and [Glu 4 ]-analogues. Chemotactic activity seemed to be correlated with the presence of the [Thr 1 ]-residue, while the [Phe 3 ]- and [D-Ala 4 ]-analogues exerted significant activity. The stimulation of cell locomotion after pre-incubation of the cells with tuftsin proved less sensitive towards variations in the amino acid sequence. All analogues stimulated chemotaxis, except the [Glu 4 ]-derivative, while random migration was stimulated by [Phe 3 ]-, [Phe 4 ]- and [D-Ser 1 ]-tuftsin. These analogue studies indicated that chemotaxis and random migration can be influenced by different chemical structures.

Defective Immune Complex Degradation by Monocytes in Patients with Systemic Lupus Erythematosus.

The binding of 125I‐labelled anti‐bovine serum albumin (BS A)‐BSA immune complexes (IC). giving a final molar antibody to antigen ratio of 1:1. to monocytes isolated from 18 patients with systemic lupus erythematosus (SLE) and from 10 normal healthy donors was quantitatively investigated. The degradation of the hound IC by the same monocytes was kinetically determined at the same time. The assays were performed on monocyte monolayers. Scatchard plots at 4°C demonstrated that monocytes from patients with active SLE expressed a mean Fey receptor (FcR.) number that was 22% higher than that of the controls, although this did not reach statistical significance. The FcR number of normal monocytes and the degradation rate of anti‐BSA‐BSA complexes by the same cells showed a positive correlation. At the same time, the digestion of anti‐BSA‐BSA complexes by monocytes of SLE patients with active disease was prolonged, despite their enhanced FcR‐ligand binding. The dissociation of FcR‐ligand binding and FcR‐mediated degradation suggests that the IC degradation is controlled by altered biochemical mechanisms in the monocytes of SLE patients.