ABSTRACT
To enhance degradation of unconjugated bilirubin in hyperbilirubinemic subjects, we synthesized a bilirubin oxidase (EC 1.3.3.5) (BO) derivative (PEGBO) by covalently linking (2,4-bis[O-methoxy(polyethyleneglycol)]-6-chloro-s-triazine) (PEG) to the enzyme. Intravenously injected BO in rats disappeared from the circulation with a half-life of 2.5 min; the half-life of PEGBO was 190 min. Intravenously injected BO minimally and transiently decreased plasma bilirubin levels in jaundiced Gunn rats and in bile-duct-ligated jaundiced rats. In contrast, PEGBO rapidly and substantially decreased plasma bilirubin levels and the effect persisted for longer than 3 h. Renal dysfunction often occurs in patients with liver diseases. To study the role of bilirubin toxicity for the kidney, functions of transtubular transport for organic anions was measured in bile-duct-ligated jaundiced animals before and after treatment with PEGBO. Bile duct ligation decreased urinary excretion of phenolsulfophthalein (PSP), an organic anion used for renal function test. Treatment of the jaundiced animals with PEGBO increased the rate of PSP disappearance from the circulation and normalized its urinary excretion. Thus, PEGBO might be useful for the study of bilirubin toxicity in jaundiced animals.
Subject(s)
Bilirubin/blood , Oxidoreductases Acting on CH-CH Group Donors , Polyethylene Glycols/blood , Triazines/blood , Animals , Bile Ducts , Half-Life , Hyperbilirubinemia/drug therapy , Immunodiffusion , Kidney/drug effects , Ligation , Liver/metabolism , Oxidoreductases/blood , Oxidoreductases/immunology , Polyethylene Glycols/chemical synthesis , Polyethylene Glycols/immunology , Polyethylene Glycols/pharmacology , Rats , Rats, Gunn , Triazines/chemical synthesis , Triazines/immunology , Triazines/pharmacologyABSTRACT
We studied the differences in protein composition and immunologic reactivity of two E. coli-derived L-asparaginase (l-Asp) preparations (I and II), Erwinia-Asp (III) and PEG-modified E. coli l-Asp (IV). On gel filtration, each of preparations I-III showed three major peaks at 100, 270 and 460 KD, all with enzyme activity, whereas PEG-Asp showed peaks at 35 and 220 KD. On SDS-PAGE one major subunit could be identified at 32 KD (I and II) or 40 KD (III), whereas PEG-modified l-Asp could only be detected by lowering the polyacrylamide concentration and gave a single band above 200 KD. Using a polyclonal rabbit antibody generated against preparation I, only the E. coli l-Asp preparations (I and II) formed precipitin lines on Ouchterlony double diffusion. After freezing and thawing, preparation IV also reacted with this antibody. In sera from patients treated with preparation I, antibodies (detected by ELISA) reacted with preparations I and II but not with preparations III and IV. These results indicate that Erwinia-Asp (III) and PEG-Asp (IV) are distinct from E. coli preparations (I and II) by molecular weight and immunological behavior. They also provide an experimental rationale for the use of Erwinia-Asp as well as PEG-Asp in E. coli Asp-sensitized patients.
Subject(s)
Asparaginase/immunology , Chromatography, Gel , Cross Reactions , Electrophoresis, Polyacrylamide Gel , Enzyme-Linked Immunosorbent Assay , Erwinia/enzymology , Escherichia coli/enzymology , Humans , Molecular Weight , Polyethylene Glycols/immunology , Precursor Cell Lymphoblastic Leukemia-Lymphoma/immunologyABSTRACT
Bilirubin oxidase (BOX) derived from Myrothecium verrucaria was modified with polyethylene glycol (PEG). When the conjugated PEG-BOX was given intravenously to rats, its plasma half-life was 20 times longer than that of native BOX. In our preliminary investigations with experimentally jaundiced rats, the plasma bilirubin level dropped to normal after only one injection, and the low bilirubin level could be maintained for 12-48 hr; native BOX had a transitory suppressive effect that lasted only a few hours. The antigenicity of PEG-BOX was greatly reduced as expected. PEG-BOX appears to have potential value for the treatment of hyperbilirubinemia observed in such diseases as fulminant hepatitis and neonatal bilirubin encephalopathy.
Subject(s)
Jaundice/drug therapy , Oxidoreductases Acting on CH-CH Group Donors , Oxidoreductases/therapeutic use , Polyethylene Glycols/therapeutic use , Animals , Antigens/immunology , Bilirubin/blood , Cholestasis/drug therapy , Epitopes , Half-Life , Hyperbilirubinemia, Hereditary/drug therapy , Immune Sera/immunology , Immunization , Immunodiffusion , Jaundice/blood , Male , Oxidoreductases/immunology , Oxidoreductases/pharmacokinetics , Polyethylene Glycols/immunology , Polyethylene Glycols/pharmacokinetics , Rats , Rats, Gunn , Rats, Inbred StrainsABSTRACT
The therapeutic effectiveness of xenogeneic monoclonal antibodies (MAbs) (xIg) or their conjugates with toxins (xIg-Tx) is undermined because of their inherent immunogenicity. This complication may be overcome by converting the antigenic xIg to tolerogenic derivatives by coupling an appropriate number of monomethoxypolyethylene glycol (mPEG) chains (MW 6400) onto an xIg molecule. In our study, the test system consisted of inbred mice and human (myeloma) monoclonal immunoglobulins (HIgG) which were used in lieu of xIg; the immunizing antigen was heat-aggregated HIgG. The results of a variety of experimental protocols demonstrate that a long-lasting suppression (greater than 95%) of anti-HIgG antibodies for periods in excess of 300 days was achieved by administration of tolerogenic HIgG(mPEG)n conjugates in spite of multiple injections of the immunizing antigen. Thus, pre-treatment of hosts with mPEG conjugates of xIg or of xIg-Tx is envisaged as a potential method for overcoming the antigenicity of these anti-tumour agents.
Subject(s)
Antibodies, Monoclonal/immunology , Immune Tolerance , Immunotoxins/immunology , Polyethylene Glycols/immunology , Animals , Female , Mice , Mice, Inbred Strains , Time FactorsABSTRACT
The specific tolerance induced in mice by conjugates of human monoclonal IgG (HIgG) with monomethoxypolyethylene glycol (mPEG) was transferred to normal mice by spleen cells or a surface immunoglobulin negative (sIg-) Lyt-2+ subpopulation of these cells. Although transferable tolerance was demonstrable 6 to 14 days after treatment of the cell donors with tolerogen, the state of tolerance persisted in the treated mice for at least 43 days. Moreover, an extract prepared by freezing and thawing of the sIg- spleen cells obtained from mice 6 days after treatment with HIgG(mPEG)20 was capable of reducing (greater than 85%) the immune response of normal mice to heat aggregated HIgG. On the basis of these results, it is suggested that similar tolerogenic mPEG derivatives of xenogeneic monoclonal immunoglobulins (XIg) may prove to be useful therapeutic agents in man when administered before treatment with the unmodified XIg.
Subject(s)
Antibodies, Monoclonal/immunology , Immune Tolerance , T-Lymphocytes/immunology , Animals , Antigens, Ly/immunology , Immunization, Passive , Immunoglobulin G/immunology , Macromolecular Substances , Mice , Polyethylene Glycols/immunology , Species Specificity , Spleen/immunology , T-Lymphocytes, Regulatory/immunology , Time FactorsABSTRACT
Conjugates of protein antigens with an optimal number of monomethoxypolyethylene glycol (mPEG) chains of an appropriate molecular weight had been shown to suppress murine IgE responses to the unmodified antigen. To investigate the possibility that the tolerogenic capacity of these mPEG conjugates is attributable to a defect in macrophage (M phi) presentation of their antigenic determinants, the activity of ovalbumin (OA)-mPEG conjugates when pulsed onto mouse peritoneal adherent cells (M phi) was compared in this study with their activity in solution. Surprisingly, in contrast to the suppressogenic capacity of mPEG conjugates in solution, the OA-mPEG pulsed M phi appeared to exert a helper effect when injected intraperitoneally (ip), i.e., after subsequent immunization with dinitrophenylated OA (DNP3-OA) in Al(OH)3, the mice showed accelerated IgE and IgG1 antibody responses to OA and DNP. However, when M phi were exposed to limiting concentrations of OA or OA-mPEG, markedly higher concentrations of OA-mPEG were required to yield pulsed M phi, exerting a significant helper effect. It was concluded that although M phi were capable of presenting the OA determinants of OA-mPEG conjugates to helper T (Th) cells, the preparations of modified antigen were presented less effectively than native OA.
Subject(s)
Antibody Formation , Antigen-Presenting Cells/immunology , Lymphocyte Cooperation , Macrophages/immunology , Polyethylene Glycols/immunology , Animals , Dose-Response Relationship, Immunologic , Immune Tolerance , Immunoglobulin E/biosynthesis , Immunoglobulin G/biosynthesis , Mice , Ovalbumin/immunologyABSTRACT
A series of new, covalent polyethylene glycol (PEG)-streptokinase adducts were synthesized and characterized. PEGs with average molecular weights of 2,000, 4,000, and 5,000 were activated with carbonyldiimidazole and coupled to the protein under standardized reaction conditions. Steady-state kinetic analysis demonstrated comparable Km values for the activation of plasminogen by streptokinase, PEG-2-streptokinase, and PEG-4-streptokinase. The kcat values were somewhat decreased when PEG-2 or PEG-4 was coupled to the streptokinase. Activation by the PEG-5 adduct did not follow Michaelis-Menten kinetics under the conditions employed in this study. Plasmin activity obtained by incubating streptokinase derivatives with plasminogen also was studied as a function of time with each of the PEG-streptokinase derivatives. By this assay, incubations containing PEG-5-streptokinase and unmodified streptokinase demonstrated comparable activity while reaction mixtures containing PEG-2-streptokinase and PEG-4-streptokinase were slightly more active. Streptokinase incubated with plasminogen at a 1:1 molar ratio was extensively degraded after 30 min whereas PEG-2-streptokinase was resistant to plasmin cleavage. The derivatized proteins were radioiodinated and incubated in plastic microtiter plates that were coated with an immunoglobulin fraction containing antibodies to streptokinase. Binding of the PEG-streptokinase adducts was decreased by greater than 95% compared with unmodified streptokinase. Plasminogen activator complexes were formed by reacting the streptokinases with human plasminogen in vitro and the clearance studied in mice. Radioiodinated plasmin in complex with the PEG-streptokinase adducts cleared at a slower rate than did plasmin complexed with unmodified streptokinase. Catabolism of the protease still occurred by a mechanism that involved reaction with alpha 2-macroglobulin as has been described for nonderivatized streptokinase-plasminogen complex (Gonias, S. L., M. Einarsson, and S. V. Pizzo, 1982, J. Clin. Invest., 70:412-423). When more extensive derivatization procedures were utilized, PEG-2-streptokinase preparations were obtained that further prolonged the clearance of complexed 125I-plasmin; however, these adducts did not uniformly retain comparable activity. It is suggested that PEG-streptokinase complexes with greatly reduced antigenicity may be useful in the treatment of thrombotic disorders.
Subject(s)
Plasminogen Activators , Polyethylene Glycols/pharmacology , Streptokinase/pharmacology , Animals , Antigens , Female , Fibrinolysin/metabolism , In Vitro Techniques , Kinetics , Metabolic Clearance Rate , Mice , Plasminogen/metabolism , Polyethylene Glycols/immunology , Streptokinase/immunology , Streptokinase/metabolismABSTRACT
Suppression of anti-L-asparaginase (anti-A-ase) IgG and IgE antibody responses was achieved in Balb/c mice with polyethylene glycol (PEG, MW, 5,000) conjugated Escherichia coli A-ase. Following the administration of the mixture of A-ase and PEG-A-ase, antibody production to A-ase was reduced. PEG-A-ase administration prior to A-ase suppressed the primary and secondary responses to A-ase antibody. The suppression could be transferred to normal mice with spleen cells from A-ase tolerant mice. The cell transfer experiment showed that the suppression was caused by suppressor T cells. Since PEG-A-ase administration failed to suppress antibody response to ovalbumin, the suppression seemed to be A-ase specific. PEG-A-ase administration also suppressed the delayed type hypersensitivity reaction. IgG and IgE antibodies to PEG or PEG-A-ase were not detected in mice immunized with PEG or PEG-A-ase in the presence of Freund's complete adjuvant or A1(OH)3, respectively.
Subject(s)
Asparaginase/immunology , Polyethylene Glycols/immunology , Animals , Antibody Formation , Antibody Specificity , Female , Immunization, Passive , Immunization, Secondary , Male , Mice , Mice, Inbred BALB C , Rats , Rats, Inbred StrainsABSTRACT
Allergens conjugated with several simple repeating polymers have reduced allergenicity in man, but large doses retain the ability to suppress ongoing allergen-specific IgE synthesis in strains of high-responder mice. To determine whether suppression of IgE antibodies could be induced in man, preliminary trials of immunologic responses to conjugates in man were carried out in ragweed hay fever patients treated with antigen E (AgE) coupled to methoxypolyethylene glycols ( MPEGs ) of 5000 and 2000 daltons, lauryloxypolyethylene glycol of 1200 daltons, and a random copolymer of D-lysine and D-glutamic acid of 69,000 daltons. In varying degrees all these conjugates had reduced allergenicity by basophil histamine release when these conjugates were compared with native AgE and could suppress IgE response in mice. Patients received one of these conjugates or native AgE in a series of subcutaneous injections and were observed for allergic reactions. The conjugates induced a lower rate of systemic reaction than native AgE but failed to induce early suppression of IgE antibodies. Instead, early rises in IgE antibody occurred in the several groups and were followed by a slow decline during a year or more that was similar to that observed with standard immunotherapy. Because the conjugates eventually caused local and systemic allergic reactions as the dose was raised, it was not possible to test the IgE-suppressive effects at doses similar to those used in mice. In contrast, rapid sustained rises in IgG antibodies occurred in all groups. The MPEG conjugates appeared to be more effective than native AgE in this regard. The reduced rate of systemic reaction and rapid rise in IgG antibody that was noted with MPEG conjugates make them worth further exploration as agents for immunotherapy.
Subject(s)
Allergens , Plant Proteins , Pollen/immunology , Rhinitis, Allergic, Seasonal/immunology , Antibody Formation , Antigen-Antibody Complex , Antigens, Plant , Desensitization, Immunologic , Humans , Immunoglobulin E/biosynthesis , Immunoglobulin G/biosynthesis , Polyethylene Glycols/immunologyABSTRACT
The immunogenic properties of the dihapten and monohapten derivatives of polyethylene glycol with different nitroaromatic groupings were studied. 2,4-dinitrophenyl, 2, 4, 6-trinitrophenyl and trinitrophenyl-ethyl groupings were used as hapten groups. The injection of monohapten compounds was found to induce the accumulation of antibody-forming cells secreting antibodies to trinitrophenyl in the spleen of normal and athymic nude mice. As early as on day 3 the number of antibody-forming cells considerably exceeded their background level, the process of B-cell activation being, to a certain extent, thymus-independent. Dihapten compounds were not immunologically active. The effect rendered by the nitroaromatic derivatives of polyethylene glycol, revealed in this study, is linked with the known capacity of polyethylene glycol to adsorb on the surface of cell membranes.
Subject(s)
Antigens/immunology , Haptens/immunology , Nitro Compounds/immunology , Nitrobenzenes/immunology , Polyethylene Glycols/immunology , Animals , Antibody-Producing Cells/drug effects , Antigens, T-Independent/immunology , Dinitrobenzenes/immunology , Dose-Response Relationship, Immunologic , Erythrocytes/drug effects , Erythrocytes/immunology , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , Mice, Nude , Molecular Weight , Trinitrobenzenes/immunologyABSTRACT
Polyethylene glycol (PEG) precipitates obtained from sera were characterized and quantitated in 20 borderline lepromatous (BL) and polar lepromatous (LL) patients with moderate to severe erythema nodosum leprosum (ENL) before and four weeks after treatment with anti-ENL drugs, e.g., cortisone, clofazimine, chloroquine, or aspirin. Although the clinical severity of ENL subsided, there was no significant clearance of material that precipitates with PEG from the circulation. These precipitates had variable anticomplementary properties.
Subject(s)
Antigen-Antibody Complex/immunology , Erythema Nodosum/immunology , Leprosy/immunology , Polyethylene Glycols/immunology , Complement System Proteins/immunology , HumansABSTRACT
Triton extracts from thymuses and thymomas from either normal subjects or MG patients specifically bind [125I] alpha-bungarotoxin (alpha Bgt). This binding is saturable (apparent Ks = 5.9 X 10(-9) M) and can be inhibited by cold alpha Bgt, acetylcholine or nicotinic agonists. These results demonstrate the presence on human thymus cells of acetylcholine receptor (ACh.R), 2.8% for normal human thymus in regard of ACh.R concentration in human muscle. Normal human thymic extracts were used as an antigen source in the radioimmunoassay for the detection of anti-ACh.R antibodies in positive MG patients' sera. 72% of these sera were also positive with the thymic antigen. The demonstration of ACh.R in human thymus brings further support to the hypothesis of intrathymic autosensitivity against ACh.R as a major factor in MG pathogenesis.
Subject(s)
Receptors, Cholinergic/immunology , Thymus Gland/cytology , Antibodies/analysis , Antigens/analysis , Bungarotoxins/metabolism , Epitopes , Humans , Myasthenia Gravis/immunology , Myasthenia Gravis/pathology , Polyethylene Glycols/immunology , Thymoma/immunology , Thymus Neoplasms/immunologyABSTRACT
The major allergen of birch pollen (BV45) was conjugated to 2-0-methoxy polyethylene glycol-4,6-dichloro-5-triazine (mPEG). Three molecular weight variants of 4,000, 6,000 and 20,000 daltons, respectively, of the activated PEG were used. The modified preparations were labelled by 125I and both the native and the radiolabelled protein conjugates were purified by gel filtration chromatography. The relative molecular weights of the purified two peaks were preliminarily estimated by high performance liquid chromatography (two populations of greater than or equal to 100,000 or 20,000 daltons). The amino acid composition of the acid hydrolysates of the three conjugated proteins indicated that 5 residues of lysine were modified by mPEG. Other charged amino acid side chains could also be bound to the activated PEG. The immunochemical properties of the copolymers were studied. The immunogenicity and antigenicity were examined by immunizing rabbits with 125I-mPEG 4,000 daltons BV45 and 125I-mPEG 20,000 daltons BV45 and subsequently crossed immunoelectrophoresis. The clearence of the radiolabelled protein showed normal pattern. A sharp decline in the radioactivity could be measured. At days 10-12, the remaining radioactivity was below 2%. Preliminary studies showed that the modified proteins were immunogenic in rabbits. The findings were demonstrated by crossed immunoelectrophoresis of the 125I-mPEG 4,000, 6,000 or 20,000 daltons BV45 and the corresponding autoradiographs. Apparently, the immunogenicity and antigenicity of the preparations were qualitatively unaltered. The allergenicity of the modified preparation was measured in vitro by RAST and RAST inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)
Subject(s)
Pollen/immunology , Amino Acids/analysis , Chromatography, Gel , Epitopes , Haptens/immunology , Immunochemistry , Iodine Radioisotopes , Polyethylene Glycols/immunology , Radioallergosorbent Test , TreesABSTRACT
Antibodies to polyethylene glycol (PEG) were analyzed in patients with various allergies and in healthy blood donors employing passive hemagglutination. In untreated allergic patients and in healthy blood donors, naturally occurring anti-PEG antibody titers between 32 and 512 were seen in 3.3 and 0.2%, respectively. During hyposensitization with monomethoxy polyethylene glycol modified ragweed extract and honey bee venom, respectively, the patients showed an anti-PEG antibody response. Titers of 32-512 were found in 50% of the patients directly after the first treatment course. After 2 years of treatment the percentage of patients with such titers declined to 28.5%. Mercaptoethanol treatment of sera indicated that the anti-PEG antibodies predominantly were of the IgM isotype. The weak IgM response found in treated patients is considered to be of no clinical significance.
Subject(s)
Antibodies/analysis , Blood Donors , Hypersensitivity/immunology , Polyethylene Glycols/immunology , Allergens/administration & dosage , Antibody Formation , Female , Haptens/pharmacology , Humans , Immunoglobulin Allotypes/immunology , Kinetics , Male , PlacebosABSTRACT
Chemically simple and physically well-defined dinitrophenyl derivatives of polyethylene oxide (DNP-PEO) can be prepared in a wide range of forms and sizes. These materials were used to investigate the molecular basis of immunogenicity and the binding of the antigens to membrane-bound receptors. Both di- and multivalent DNP-PEO activate normal murine B lymphocytes to yield primary anti-DNP antibody response in vitro. The immunogenicity is dependent on the carrier chain length but independent of T cells. Responses comparable to those induced by DNP-conjugated polymerized flagellin are induced by divalent linear materials of medium molecular weights of about 60,000. A highly multivalent material is moderately immunogenic, but at much lower antigen doses than divalent materials. The carrier PEO does not affect B-cell responses to DNP-PEO or T-cell response to succinyl concanavalin A. Moreover, it shows no polyclonal mitogenicity at concentrations as high as 1 mg/ml. Studies of antigen binding to cell surface DNP receptors show that the strongly immunogenic materials of medium molecular weights have an appreciable tendency to bind bivalently and thus potentially to crosslink receptors. The binding of smaller, less immunogenic antigen appears predominantly monovalent.
Subject(s)
Antigens, T-Independent/immunology , B-Lymphocytes/immunology , Dinitrobenzenes/immunology , Hemolytic Plaque Technique , Nitrobenzenes/immunology , Polyethylene Glycols/immunology , Animals , Antibody-Producing Cells/immunology , Antigens, T-Independent/administration & dosage , Binding Sites, Antibody , Cytotoxicity Tests, Immunologic , Dose-Response Relationship, Immunologic , Female , Macromolecular Substances , Mice , Molecular Weight , Perissodactyla , Rosette FormationABSTRACT
Antibodies to polyethylene glycol (PEG) were raised in rabbits by immunization with monomethoxy polyethylene glycol modified ovalbumin (OA), bovine superoxide dismutase (SOD), and ragweed pollen extract (Rag), given in Freund's complete adjuvant (FCA). Immunogenicity depended on the nature of the protein and the degree of modification. With modified OA, in the presence of FCA, the majority of animals showed an anti-PEG response. With modified SOD and Rag only a small proportion of animals responded. In the absence of FCA, modified OA, given s.c., did not elicit any anti-PEG antibody response in rabbits and only a weak response in mice. PEG of MW 10,000 and 100,000 given in FCA was found nonimmunogenic in rabbits, and PEG of MW 5.9 X 10(6), given s.c. to mice, showed no or very poor immunogenic properties. Gel diffusion, heterologous passive anaphylaxis and passive hemagglutination were used to demonstrate anti-PEG antibodies raised to PEG-modified proteins. Specificity was confirmed by hapten inhibition of precipitation, inhibition of passive hemagglutination and cross-reactivity tests. PEG of MW greater than or equal to 4,000 produced specific precipitates, smaller molecules acted as monovalent haptens. From hapten inhibition of precipitation by PEG of MW 300 it appears that the antigenic determinant of PEG may be a sequence of 6-7 -CH2CH2O-units. Anti-PEG antibodies can be used analytically. By gel diffusion, Peg was detected in minimal concentrations of 0.1-1 microgram/ml. The clinical relevance of these findings with regard to therapy with PEG-modified enzymes and allergens in humans remains to be established.
Subject(s)
Immunization/methods , Ovalbumin/immunology , Polyethylene Glycols/immunology , Animals , Antibody Formation , Cattle , Cross Reactions , Dose-Response Relationship, Immunologic , Hemagglutination Tests , Immunodiffusion , Injections, Intramuscular , Mice , Mice, Inbred Strains , Passive Cutaneous Anaphylaxis , Polyethylene Glycols/administration & dosage , Polyethylene Glycols/pharmacology , RabbitsABSTRACT
Antibody responses of the IgE isotype were raised in mice with honey-bee venom (HBV) administered in alum or by daily injections without adjuvant. The sensitized mice were treated with single injections of HBV modified with monomethoxy-polyethylene glycol. By such treatment with modified but not with natural HBV, suppression of the IgE antibody responses was achieved. The IgG antibody responses, in contrast, were unchanged or enhanced.
Subject(s)
Antibody Formation , Bee Venoms/immunology , Polyethylene Glycols/immunology , Adjuvants, Immunologic/immunology , Animals , Antibodies, Anti-Idiotypic/immunology , Dose-Response Relationship, Immunologic , Immunization , Immunoglobulin E/immunology , Immunoglobulin G/immunology , Mice , Mice, Inbred CBAABSTRACT
During solubilization of immune complexes C3b becomes fixed to the immunoglobulin part and serves as a receptor for the alternative complement pathway control protein H. The H-C3b immune complex interaction can be made detectable using 4% polyethyleneglycol to separate free from bound 125I-H. Tetanus toxoid (Te)/anti-Te complexes kept soluble with fresh serum and containing 125 IU of specific antibody bound 18% of 125I-H; when fresh serum was chelated with 10 mM EDTA, 125I-H binding was only 5%. On sucrose density gradients, the H-binding material sedimented in the range of 12 to 30 S. In 36 serum samples from rheumatoid arthritis (RA) patients and in 12 serum samples from patients with systemic lupus erythematosus (SLE), 125I-H binding was significantly elevated to 9.5 +/- 4.7% (mean +/- 1 SD) and 13.3 +/- 5.6%, respectively, while 125I-H binding by 36 normal human sera was 4 +/- 2%. RA samples (17/36, 47%) and SLE samples (9/12, 75%) had H-binding values increased by more than 2 SD above the normal mean. The serum samples were also assessed for conglutinin- and C1q-binding activities; a significant correlation between H and C1q binding was observed (P less than 0.001); there was no correlation between H and conglutinin binding. Although binding to immune complexes through its interaction with C3b, H clearly detects a population of complexes other than conglutinin, thus expanding the possibilities of further characterizing pathological complexes.
Subject(s)
Antigen-Antibody Complex/immunology , Complement Activation , Complement C3b Inactivator Proteins/immunology , Complement Pathway, Alternative , Antigen-Antibody Complex/analysis , Complement Factor H , Humans , Iodine Radioisotopes , Polyethylene Glycols/immunology , Tetanus Antitoxin/immunology , Tetanus Toxoid/immunologyABSTRACT
Some of the possible mechanisms by which polyethylene glycol (PEG) augments the ability of MOPC-315 tumor bearer spleen cells to mediate in vitro antitumor cytotoxicity were evaluated. The level of antitumor cytotoxicity obtained in 5-day cultures of tumor bearer spleen cell suspensions correlated inversely with the percentage of Trinitrophenol (TNP)-rosettable cells (presumably metastatic tumor cells) present in the spleen. The kinetics of decrease in the percentage of TNP-rosettable cells coincided with the appearance of antitumor cytotoxicity. In addition, PEG was shown to interfere with the ability of viable tumor cells to suppress the in vitro generation of antitumor cytotoxicity in normal spleen cells cultured with mitomycin C-treated tumor cells. However, the decrease in the content of TNP-rosettable cells and the concurrent increase in the level of antitumor cytotoxicity were not due to direct cytotoxic and/or cytostatic effects of PEG on tumor cells. Spleen cells cultured in the presence of PEG had an increased rate of [3H]thymidine incorporation and proliferation compared to spleen cells cultured in the absence of PEG. However, the PEG-induced decrease in the percentage of TNP-rosettable cells either preceded or occurred at the same time that the PEG-induced increase in spleen cell number was observed. Therefore, spleen cell proliferation can at best explain only partially the PEG-induced decrease in the content of TNP-rosettable cells, and other mechanisms for the decrease must be considered.