Complement-immunoglobulin interactions.

Normal circulating immunoglobulin may control complement binding to targets and thereby the manifestations of autoimmune disease. Molecular analysis of IgG and IgM mutants suggests that C1q binding by IgG utilizes a core Glu-X-Lys-X-Lys motif (where X is any amino acid). Additional amino acids, particularly homologous proline residues at position 331 in IgG and 436 in IgM, appear critical for classical pathway initiation. Glycosylation of IgG heavy chain is important in C1q binding, as well as glycosylation of IgA heavy chain for alternative pathway initiation. Additional recent evidence suggests an important role for C3 in antigen presentation. The data also raise the possibility that C3 plays a significant role in the intracellular antigen processing pathway.

Immunoglobulin in the control of complement action.

Complement is a critical element of innate immunity, protecting individuals from a wide variety of microbial infections. This group of proteins is responsible for many features of inflammation and tissue damage. Because of its ability to mediate autoimmune tissue damage and to destroy host tissues, it is under tight regulation with many circulating and cell-membrane-bound complement regulatory proteins. The function of much of the circulating immunoglobulin has never been defined. We have advanced the hypothesis that one function of circulating immunoglobulin is to down-regulate complement attack on host tissues in the presence of anti-self antibody. The data to support this hypothesis are reviewed. The data are consistent with the suggestion that one mechanism of action of intravenous immunoglobulin, used to treat patients with a variety of autoimmune diseases, is prevention of complement-mediated attack on host tissues.

A laser nephelometric method for measuring alternative pathway complement activity of human and murine serum.

A kinetic method for the evaluation of alternative pathway complement activity in man and mice is presented. A laser nephelometer was employed for detection of non-sensitized rabbit erythrocyte lysis based on the observation that the intensity of laser scatter (LS) is proportional to the number of erythrocytes in suspension. During erythrocyte lysis a continuous decline in LS is observed since lytic products do not evoke LS. Utilizing the indirect Coombs test and cross-electrophoresis it was shown that rabbit erythrocyte lysis causes activation of the alternative complement pathway. This method is modified for room temperature, is independent of sample hemoglobin content and, in its micro version, it can be done with 10 microliter of human serum, i.e. 50 microliters of murine serum.

Antibodies against rabbit red blood cells in murine serum and their effect on the activity of complement alternative pathway.

Antibody content against rabbit red blood cells (anti-RaRBC) in murine sera of different strains (Swiss, CBA, C57BL/6, AKR, BALB/c) and activity of complement alternative pathway (AP) were investigated. In contrast to the CBA and C57BL/6, random-bred Swiss strain and inbred BALB/c and AKR strains are good producers of these natural antibodies. There is no correlation between AP activity and anti-RaRBC content. Isolated human anti-RaRBC antibodies, IgM and IgG classes, lead to the enhancement of APhu and APmo activity, contrary to the murine anti-RaRBC which belong solely to IgM class, and do not express this capability.

Salivary IgA secretion rate in young and elderly persons.

Immunoglobulin A (IgA) is the dominant immunoglobulin isotype on all mucosal surfaces where it acts as a first line of defense against microbial invasion. Recent investigations suggest that secretory IgA (sIgA) concentrations vary over the day due to a range of variables including dietary factors, daily mood, and exercise. In this study, salivary IgA was determined by ELISA in samples of 48 persons grouped as "young" (20-30 years old) and "elderly" (60-80 years old). Unstimulated, stabilized morning and afternoon saliva was collected during 7 consecutive days. Saliva flow, total proteins, sIgA concentrations, and sIgA secretion rates were determined. The main finding was that saliva flow and sIgA secretion rate were significantly lower in the elderly than in the young. Salivary IgA secretion rate was found to be independent of total proteins secreted in all samples. There is individual variability within a particular age group. It was also found that stress and daily events influence the sIgA secretion rate.

Production and characterization of the monoclonal antibody against SGP120, a novel serum protein.

A monoclonal antibody designated B1.9.E-2 was produced and characterized to facilitate study of the immunizing antigen-a serum glycoprotein of 120 kDa (sgp 120) of unknown function. The antibody, produced in mice, reacts with 1-2 epitopes located in the N-terminal region of the sgp 120 molecule. The affinity of the reaction, as determined by Scatchard analysis, is Ka = 1.13 x 10(10) I/M and is of a hydrophobic nature. The monoclonal antibody can be purified to a high degree by a modified caprylic acid method and by protein G FPLC chromatography column. B1.9.E-2 does not trigger the complement cascade, as determined by C3 RIA and immune complex solubilization assays. Both affinity purified (C4b Sepharose) and chromatographically isolated (using jacqualine agarose) sgp 120 were recognized by B1.9.E-2. The monoclonal antibody can be utilized for affinity purification of sgp 120, for detection of intact or cleaved sgp 120 in biological samples of healthy and ill individuals, and for the number of functional and neutralization assays aimed at resolving the physiologic role of sgp 120.

[Isoelectric spectrum of IgD in myeloma]. / Izoelektricni spektri mijelomskih IgD.

Isolated and highly purified myeloma IgD DEK, STA and SAR were subjected to isoelectric focusing in thin layer of polyacrylamide gel using equipment and PAG plate 3, 5-10 from LKB. Although homogeneous in electrophoresis on cellulose acetate folien, immunoelectrophoresis and DISC PAA gel electrophoresis analysed IgD showed high isoelectric heterogeneity. They formed isoelectric spectra with 24-27 pl zonnes ower the pH range 5,4-9,3. Based on densitometric analysis of gel stripps zonnes with a small protein content were excluded from calculation of the real isoelectrical range. According to that manipulation isoelectrical range was determined as pH 6-8. Heterogeneity of isolated myeloma IgD may be due to the post-synthetic transformation of molecules in vivo as well to degradation and/or aggregation of IgD in vitro during the preparation of samples for isoelectrofocusing. However, myeloma IgD are in fact more heterogeneous in isoelectrofocusing than myeloma immunoglobulins of other classes.

Complement activation in stored platelet concentrates.

Activation of platelets during preparation and/or storage of platelet concentrates in plastic containers at room temperature has recently been recognized. Many different biologic causes of this activation have been postulated. Activated complement, as a multi-enzyme system, is one of the possible sources of molecules leading to platelet activation. To detect complement activation, functional complement activity and the generation of complement-derived ligands were investigated in platelet concentrate supernatant plasma during 5 days of storage at room temperature. Hemolytic tests for functional classical and alternative pathway activity were used, as was the kinetic test for complement-mediated inhibition of immune complex precipitation. The presence of C3 activation products (C3, C3c, C3dg) was investigated in plasma by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blotting procedures and on platelets by immunofluorescence. Activation of complement was evident during storage, and C3c and C3d fragments were clearly demonstrated in plasma. The amount of C3d fragments on platelets gradually rose during the first 3 days of storage. At the end of 5 days of storage, the platelets became C3d negative. There are two possible mechanisms of C3d disappearance--shedding and/or further degradation of C3d fragments. Those results indicated that complement activation and the generation of complement-dependent ligand-receptor interaction may be mechanisms for platelet activation in concentrates stored at room temperature.

Study of complement effects on kinetics of immune precipitation.

Influence of complement on the kinetics of immune precipitation was investigated by using simple kinetic tests. In the early phase of immune precipitate (IPPT) formation, it was demonstrated that complement caused inhibition of precipitation only. After formation of a certain critical IPPT mass, alternative pathway-dependent enhancement of precipitation was observed, followed by solubilization of 'enhanced' IPPT and, at last, by solubilization of preformed IPPT. Contrary to inhibition and IPPT solubilization, enhancement of IPPT formation does not change IPPT ability to react with fresh complement.

[Isolation and determination of the molecular mass of the RHoD antigen in human erythrocytes]. / Izolovanje i odredivanje molekulske mase RhoD antigena humanih eritrocita.

RhoD antigen was isolated from human erythrocytes using membrane solubilization by 2-mercaptoethanol and nonionic detergents BRIJ 35. Initial treatment of membrane using water at pH 12, failed to solubilize D antigen, which proved that D antigen is an integral protein of erythrocyte membrane. D antigen molecule mass was investigated by ultrafiltration and gelfiltration. Molecular mass was determined by ultrafiltration in the range of 10-20 000 daltons, and by gelfiltration on Sephacryl S--200 as 32 700. Using gelfiltration on Sephadex G-25, D antigen molecular mass was determined to be less than 25 000 and was approximately 14 000 daltons. Discrepancy between those two ways of determination by gelfiltration and ultrafiltration could be explained by the possibility that D antigen globularity is practically unexpressed.

Kinetic study of rheumatoid factor influence on complement-mediated modulation of immune precipitation.

Human monoclonal IgM kappa rheumatoid factor PA (mRF) expressed various effects on complement-dependent modulation of ovalbumin/antiovalbumin lattice formation measured kinetically by laser light scattering (LLS): (1) in the absence of complement, mRF (44.2-177 U/ml) caused enhancement of LLS in a dose-dependent manner; (2) mRF partially prevented expression of complement-mediated inhibition of lattice formation and, at the same time, complement inhibits mRF-dependent LLS enhancement; (3) complement caused disruption of lattice-mRF bonds during solubilization of preformed lattice-mRF complex as demonstrated by specific mRF activity in supernatants. This effect is dependent on complement activity and on both complement and mRF concentrations; (4) ovalbumin/antiovalbumin complexes gradually lost the ability to react with mRF during the first 2 min of complement-mediated inhibition of lattice formation.

Regulation of complement activity by immunoglobulin. I. Effect of immunoglobulin isotype on C4 uptake on antibody-sensitized sheep erythrocytes and solid phase immune complexes.

Intravenous Ig, composed principally of IgG, prevents complement attack by inhibiting C3 and C4 uptake onto target cells and tissues. Using two different models, Ab-sensitized SRBC and BSA-anti BSA solid phase immune complexes, we have examined the complement inhibitory capacity of three Ig classes (IgG, IgM, IgA) focussing on inhibition of C4 uptake. It was found that on both a weight and molar basis, monomeric serum IgA and IgM were far more active than IgG (weight efficiency ratios were 1.0, 20.8, and 236.3, and molar efficiency ratios 1.0, 24.0, and 1382.9 for polyclonal IgG, IgA1, and IgM, respectively). Monoclonal IgM were less active than polyclonal IgM (50% inhibition was achieved in SRBC model by 0.022, 0.30, 1.6, and 1.6 mg/ml of polyclonal IgM and monoclonal IgM from patients Lew, Will, and Pri). Secretory IgA was less active than serum IgA1 and similar in inhibitory activity to IgG (weight and molar efficiency ratios 1.5 and 0.6 compared with IgG). All tested preparations were less active in the solid phase immune complex model than in the sensitized cell model. A mixture of Igs of different isotypes was somewhat more active than any isotype alone. These results suggest that polyclonal serum IgA and IgM can also be considered for active therapy in diseases accompanied by the activation of classical complement pathway.

[Anti-RaRBC antibodies in human serum. I. The presence of complete anti-RaRBC antibodies in the serum of healthy persons and their isotype specificity]. / Anti-RaRBC antitela u serumu coveka. I. Prisustvo kompletnih anti-RaRBC antitela u serumu zdravih osoba i njihova izotipska specificnost.

Agglutinins against rabbit erythrocytes (RaRBC) were detected in 60 investigated normal human ser in titres ranging 1:20 - 1:1280. Titres of saline anti-RaRBC were not dependent to temperature nor to the blood groups of donor sera. Saline agglutinins were identified as antibodies of IgM class based on the result of gelfiltration and absorptions on anion exchanger and SpA-Sepharose immunoadsorbent as well as on mercaptoethanol and sulfitolysis sensitivity. IgG anti-RaRBC antibodies incapable to agglutinate RaRBC in saline were detected using indirect Coombs test in the second protein peak of gelfiltration. Some characteristics of anti-RaRBC saline agglutinins were compared with human natural isohemagglutinins.

[Anti-RaRBC antibodies in human serum. II. Presence of incomplete anti-RaRBC antibodies in the serum of healthy persons]. / Anti-RaRBC antitela u serumu coveka. II. Prisustvo inkompletnih anti-RaRBC antitela u serumu zdravih osoba.

Non-agglutinating character of some anti-rabbit erythrocytes (RaRBC) antibodies was demonstrated. Non-agglutinating ("incomplete") antibodies were identified mainly as IgG but in some sera were detected also in IgA and IgD class using indirect Coombs test with monospecific antisera. Neutralization and separation of "complete" (IgM) antibodies were performed using 2-mercaptoethanol treatment and/or preparative immunochemical techniques. Some kind of restriction to kappa light chains and to IgG2 subclass has also been observed but monoclonal character of "incomplete" antibody was excluded. Specificity of reaction of IgG anti-RaRBC antibodies with RaRBC membrane antigens was documented using multiple adsorption-identification experiments. Non-specific (Fc-mediated) reaction was excluded in contrast to previously published results on the presence of Fc receptor on RaRBC.

Identification of gliadin presence in pharmaceutical products.

Celiac disease is characterized by hypersensitivity to the alcohol-soluble wheat proteins called gliadins. Total avoidance of gliadin is the lifelong treatment for such patients. However, wheat gliadins are often present as impurities in industrial starch commonly used in the preparation of different pharmaceutical products. Therefore, some drugs might contain gliadin, which can be dangerous for sensitive patients if taken in large amounts or used permanently. The purpose of this study was to develop a sensitive, reliable assay that is specific for the detection of gliadins in pharmaceutical products. Gliadins were extracted here using sodium dodecyl sulfate rather than 70% ethyl alcohol, which has been the traditional solvent. This gliadin extract was utilized in a dot-blot assay that incorporated an antigliadin antibody developed in rabbit and labeled with peroxidase. 4-Chloro-1-naphthol was used as a peroxidase-specific substrate. Isolated wheat gliadin was used as the positive control. Dilution experiments showed that the lower level of sensitivity for the assay was in the range of 0.0045 mg/ml of gliadin, which is a concentration level lower than that suggested for a gluten-free diet. The assay developed here revealed that 71.2% of 59 prescription and nonprescription drugs tested contained gliadin in the amount detected by our dot-blot assay. The prescription drugs tested were among the top 50 most frequently dispensed in U.S. community pharmacies. The nonprescription drugs were among those that constitute the largest sales in the United States. The results showed that the simple dot-blot assay developed here can be used for pharmaceutical testing performed either by hospital laboratories or by patients themselves.

[Preparation of human albumin preparations]. / Pripremanje humanih albuminskih preparata.

Structure, physical and biochemical characteristics of albumin have already been investigated and described in full detail. Preparation of albumin solution preparations of various concentrations for human application started more than 40 years ago. Person deserving most credit for it is E. Cohn, who first used ethyl alcohol on low temperatures for the separation of certain fractions among which albumin took the largest part. Many modifications of Cohn's method are used today, though the most frequently used one is modification by Kistler and Nitschman. Some new methods appeared recently, ensuring higher degree of economy and efficacy, better yield of albumin and higher purity of final preparations. One of those methods--chromatography method--is recommended by Swedish authors (J. Curling et al.). Thanks to its multiple physiological functions and stability, albumin takes very important place in therapy of many diseases, both in peace and in states of emergency.

The therapeutic efficacy of VBCMP-M2 protocol in multiple myeloma.

Within a period from March 1983 to November 1988, 93 newly diagnosed patients with multiple myeloma, 47 men and 46 women aged from 41 to 80 years were treated with protocol VBCMP-M2. A response was achieved in 30 (60%) patients in stage II and 22 (51%) in stage III, the overall response rate being 56%. 41% of all patients entered plateau phase, more frequently in stage II (46%) than in stage III (37%) patients. The median survival of all patients was 35 months. Comparing these groups of patients with other series from the literature, we were not able to demonstrate any advantage of this multidrug combination over other combinations or even melphalan and prednisone alone.

Primary bile acid malabsorption. Histologic and immunologic study in three patients.

Three patients are presented with a history of chronic watery diarrhea due to bile acid malabsorption, proved by the tauro-23[75Se]selena-25 homocholic acid test and an unequivocal response to cholestyramine therapy. Fecal fat tests, Schilling tests, and barium studies of the small intestine and colon were all normal. Jejunal biopsies were normal but multiple biopsies of the terminal ileum, performed by retrograde ileoscopy, showed uniform crypt hyperplastic villous atrophy and features of colonic metaplasia with increased mononuclear infiltration of the lamina propria. All 3 patients demonstrated evidence of abnormal immune function, including the presence of serum autoantibodies, circulating immune complexes, and hypocomplementemia. One patient had Sjögren's syndrome. This disorder, which might be immunologically mediated, should be called primary bile acid malabsorption and should be distinguished from other ileal disorders.