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Biomedical subjects

L F Fries

Publications and source records attributed to L F Fries.

At least 37 records · Page 2Linked to original sources
PubMed1990

Comparison of live attenuated cold-adapted and avian-human influenza A/Bethesda/85 (H3N2) reassortant virus vaccines in infants and children.

Randomized, placebo-controlled studies with 10(3)-10(7) 50% tissue-culture infectious dose (TCID50) of avian-human (ah) and cold-adapted (ca) influenza A/Bethesda/85 (H3N2) reassortant viruses were completed in 106 seronegative young children 6-48 months of age. Although the reassortants differed in six of eight RNA segments, they exhibited similar properties in level of attenuation, infectivity, immunogenicity, and efficacy. The 50% human infectious dose was 10(4.6) TCID50 for ah and 10(4.4) for ca vaccines. Both reassortants were satisfactorily attenuated with restricted replication and were no more reactogenic than placebo. The mean peak titer of virus shed was 10(1.5) (ah) to 10(2.0) (ca) TCID50/ml, and each of 37 isolates tested retained their characteristic vaccine phenotypes. Infection with ah or ca virus conferred immunity to experimental challenge with homologous virus. These findings indicate that both ah and ca influenza A/Bethesda/85 (H3N2) reassortants should be suitable vaccine candidates for use in healthy infants and young children.

Antibodies, Viral↗
PubMed1989

A new simplified procedure for C1 inhibitor purification. A novel use for jacalin-agarose.

C1 inhibitor (C1-INH), the major regulatory protein of the classical pathway of complement activation, is also involved in the regulation of several other plasma proteolytic systems including the coagulation, fibrinolytic and contact systems. All the previously published methods for the purification of C1-INH are time-consuming and some do not yield highly pure protein. Recently, it was reported that Jack fruit (Artocarpus integrifolia) lectin, also called jacalin, binds C1-INH. Since jacalin binds only a small number of human serum proteins it appeared that jacalin-agarose affinity chromatography would constitute a very selective early step for the purification of C1-INH. Consequently we have designed a new, simplified three-step procedure for the purification of C1-INH which includes PEG fractionation, jacalin-agarose chromatography and hydrophobic interaction chromatography on phenyl-Sepharose which takes advantage of the marked hydrophilicity of the inhibitor. This procedure has three major advantages over those which have been the most frequently used. Firstly, it includes only two fast chromatographic steps. Secondly, because the C1-INH pool is cleanly and predictably separated from the unwanted proteins by differential elution conditions in both chromatographic steps, no antigenic or functional assays are required to define the desired peaks. Thirdly, only the final product is dialyzed while all other methods required several buffer changes. For these reasons this procedure is much faster and simpler than the previously published methods. About 10-12 mg of highly purified and fully active C1-INH can be obtained within 1 day from 120 ml of plasma giving an average yield of 40-45%. This method may thus be highly adaptable to bulk purification for clinical use or for preparation of genetically or pathologically altered C1-INH from clinical specimens.

Blotting, Western↗
PubMed1989

Complement abnormalities in multiple myeloma.

PURPOSE: Patients with multiple myeloma have been shown to have defective opsonization and C3 deposition. Previous studies have suggested that defective C3 deposition may be related to a failure of C3 activation in myeloma serum, the mechanism of which is unknown. We therefore decided to investigate the underlying mechanism responsible for the failure in C3 activation and deposition. PATIENTS AND METHODS: The study consisted of 10 patients from whom a total of 12 serum specimens were obtained. Normal serum was prepared from a pool of serum specimens in four healthy male donors. We evaluated, in vitro, the kinetics of C3 deposition onto zymosan using radiolabeled C3 under various conditions. We also measured the serum levels of a variety of complement components using standard methods. RESULTS: Five of 10 patients' sera demonstrated poor C3 deposition onto zymosan at all time points, whereas an additional two showed poor C3 deposition at early time points but a rebound to normal by 30 minutes. Multiple components of the classical and alternative complement pathways were decreased in many patients, with the most striking abnormalities occurring in those with the poorest C3 deposition. No single complement component abnormality was found to be common to the group. Elevations in Bb fragment concentration strongly suggest in vivo activation as the likely mechanism for depletion of alternative pathway components; the mechanism for classical pathway abnormalities is less clear. There was an inverse correlation between paraprotein concentration and abnormal C3 deposition (p less than 0.0001) and C3 (p less than 0.0005) and C4 (p less than 0.0001) concentrations. However, no consistent evidence of fluid-phase complement consumption was present. CONCLUSION: The defect in C3 activation and deposition in multiple myeloma cannot be explained on the basis of a single complement component abnormality but rather is due to a heterogeneous group of complement abnormalities. Although no correlation between in vitro abnormalities and clinical status was identified in this small group of patients, it is likely that the described complement defects play an important role in defective host defense in multiple myeloma.

Complement Activation↗
PubMed1989

Mechanism of therapeutic effect of high-dose intravenous immunoglobulin. Attenuation of acute, complement-dependent immune damage in a guinea pig model.

Studies were performed in in vitro and in vivo models to assess the effect of intravenous immunoglobulin (IVIG) on the development of acute complement-mediated tissue damage. IVIG significantly increased the duration of survival and frequently prevented the death of guinea pigs injected with anti-Forssman antiserum to cause lethal Forssman shock; no control animal treated with albumin and/or maltose vehicle survived. The most pronounced effect was achieve by delivering IVIG as one slow injection at 1,800 mg/kg 3 h before Forssman shock was elicited. Infusion of guinea pig IgG at the same dosage was similarly protective. A strong positive correlation was found between IgG plasma levels and survival time in guinea pigs treated with graded doses of IVIG. Therapy itself did not affect C3 and C4 levels nor the capacity to activate these components. In vitro studies showed almost complete inhibition of C3 uptake onto IgG-sensitized erythrocytes using serum from an IVIG-treated animal. We suggest that supraphysiologic levels of IVIG act in part by preventing active C3 fragments from binding to target cells. Infusion of high dose IVIG may be a rational approach to modulating acute, complement-dependent tissue damage in a variety of diseases in man.

Animals↗
PubMed1989

High-dose intravenous immunoglobulin modifies complement-mediated in vivo clearance.

The mechanism of effect of high-dose intravenous immunoglobulin (IVIG) therapy in immune cytopenias is incompletely known. One of the leading theories ascribes the short-term effects of IVIG to the competition of infused IVIG for Fc receptors, thereby inhibiting IgG-mediated clearance. Using a system independent of IgG-Fc receptor interactions, we examined another potential mechanism of IVIG action. Guinea pigs were infused with a human IVIG preparation at 600 mg/kg/day for two consecutive days. Parallel groups of animals were treated with the same volume and/or concentration of saline and albumin. Clearance of IgM-sensitized guinea pig erythrocytes, which is wholly complement dependent, was significantly retarded in animals treated with high-dose IVIG. The effect was specific for IVIG, since human albumin (as a second foreign protein) failed to change the clearance of IgM-sensitized guinea pig erythrocytes. Experiments in which IVIG-treated animals were subjected to pre- and posttreatment clearance studies revealed heterogeneity among individual animals in respect to their response to IVIG infusions. Decrease of available plasma complement components did not account for the effect, since both C3 and CH50 values remained unchanged after IVIG treatment, despite rising levels of IVIG in sera of treated animals. The results of in vitro C3 uptake studies and the effect of IVIG on clearance of preopsonized cells suggest that IVIG produces a kinetic depression of C3 uptake and modifies the process of complement fragment deposition on erythrocytes. A generalized effect on mononuclear phagocytes is less likely but cannot be wholly ruled out. These studies establish another potential mechanism of IVIG action and suggest extension of its use to other complement-mediated diseases.

Animals↗
PubMed1988

Acquired angioedema: observations on the mechanism of action of autoantibodies directed against C1 esterase inhibitor.

We describe a mechanism of action of autoantibodies reactive with the C1 esterase inhibitor (C1EI) molecule found in three patients with acquired angioedema without associated diseases. All of these patients have a circulating C1EI of lower molecular weight than that of normal control subjects. When native C1EI was added to patient, but not control, plasmas, it was cleaved to a lower molecular weight fragment under conditions that allowed contact system activation. In a partially purified system, patient immunoglobulin preparations impaired stable complex formation between plasmin and C1EI and exaggerated the cleavage of the latter to a lower molecular weight fragment. We propose that the autoantibody does not interfere with the cleavage of the bait sequence of the inhibitor but reduces the availability of the reactive center of the molecule in a way that interferes with the irreversible inhibition of target enzymes. In this way unregulated activation of the kinin and complement pathways occurs, leading to disease manifestations via as yet uncharacterized mediators.

Angioedema↗
PubMed1988

C3b receptor activity on transfected cells expressing glycoprotein C of herpes simplex virus types 1 and 2.

Glycoprotein C from herpes simplex virus type 1 (gC-1 from HSV-1) acts as a receptor for the C3b fragment of the third component of complement on HSV-1-infected cell surfaces. Direct binding assays with purified gC-1 and C3b demonstrate that other viral and cellular proteins are not required for this interaction. Although C3b receptor activity is not expressed on HSV-2-infected cell surfaces, purified gC-2 specifically binds C3b in direct binding assays, suggesting that gC-1 and gC-2 are functionally similar. Here, we used a transient transfection system to further characterize the role of gC-1 and gC-2 as C3b receptors and to localize the site(s) on gC involved in C3b binding. The genes for gC-1 and gC-2 were each cloned into a eucaryotic expression vector containing the Rous sarcoma virus long terminal repeat as the promoter and transfected into NIH 3T3 cells. The expressed proteins were similar in molecular size, extent of carbohydrate processing, and antigenic properties to gC-1 and gC-2 purified from infected cells. Using a double-label immunofluorescence assay, we found that both gC-1 and gC-2 were expressed on the surfaces of transfected cells and bound C3b. These results suggest that other proteins expressed during HSV-2 infection prevent receptor activity. We constructed three in-frame deletion mutants of gC-2 to identify domains on the protein important for C3b receptor activity. These mutants lacked amino acids 26 to 73, 219 to 244, or 318 to 346. The mutant protein lacking residues 26 to 73 was reactive with two monoclonal antibodies recognizing distinct epitopes, showed a wild-type pattern of carbohydrate processing, and bound C3b on the transfected cell surface. These results suggest that residues 26 to 73 are not involved in C3b binding. The other two mutant proteins were present on the cell surface, but did not bind C3b. In addition, these mutant proteins showed altered patterns of carbohydrate processing, formed aggregates, and were no longer recognized by the monoclonal antibodies. These properties indicate that removal of residues 219 to 244 or 318 to 346 disrupted the native conformation of gC-2, possibly owing to an alteration in the spacing between critical cysteine residues.

Antibodies, Monoclonal↗
PubMed1988

Interactions of a nonneutralizing IgM antibody and complement in parainfluenza virus neutralization.

While many viruses activate the complement cascade directly, this is generally not a neutralizing event in the absence of antibody. We used a nonneutralizing IgM monoclonal antibody to parainfluenza virus type 3 (PIV3) hemagglutinin-neuraminidase (HN) to explore the role of antibody in complement-dependent neutralization of PIV3. Neither the antibody nor nonimmune guinea pig serum (GPS) neutralized PIV3 significantly, but a more than 100-fold reduction in titer was found when antibody and GPS were combined. Heat-inactivated GPS or GPS lacking either of two different complement proteins were all inactive with or without antibody. Specific repletion of the deficient sera with highly purified complement proteins restored neutralizing activity, indicating an absolute requirement for the classical pathway of complement activation and lytic terminal complement components, and viral lysis was confirmed by electron microscopy. The presence of antibody before complement activation was essential; later addition had no effect. Spontaneous complement activation by PIV3 occurred via the classical pathway in the absence of antibody. Addition of antibody did not alter the overall rate or extent of complement component C3 binding to PIV3 in these experiments. We conclude that certain nonneutralizing antibodies may support complement-dependent PIV3 neutralization by facilitating viral lysis. This process does not, however, involve enhanced activation through the C3 step. Lysis may require antibody-dependent localization of the membrane attack complex or reorganization of the viral envelope structures to facilitate attack complex insertion and lysis.

Antibodies, Monoclonal↗
PubMed1988

CR1-receptor recycling in phorbol ester-activated polymorphonuclear leucocytes.

Complement-receptor type 1, CR1, which recognizes the C3b cleavage fragment of C3, is present on the membranes of human phagocytic cells, but does not mediate phagocytosis or undergo internalization unless activated by one of a variety of stimuli. Among these stimuli low doses of phorbol esters have been shown to induce a consistently increased expression of CR1, despite apparently continuous receptor internalization. We have studied the fate of internalized receptor-ligand complexes in neutrophils activated with low concentrations of phorbol dibutyrate. In our studies, we followed CR1 with either 125I-C3b, the physiologic ligand, or with 125I-Fab fragments of a monoclonal anti-CR1 antibody. We observed rapid internalization of CR1-C3b complexes by PMN treated with 10 ng/ml (1.98 x 10(-8)M) PDBu, consistent in rate and extent with previously reported results using monoclonal antibodies. The fate of the internalized ligand was studied after elution of cell-surface C3b at 0 degrees. Intracellular ligand was externalized in a time- and temperature-dependent fashion, reaching a plateau at 10-15 min. Released C3b was totally TCA precipitable and structurally unaltered, as determined by SDS-PAGE, suggesting that recycling occurs via a prelysosomal predegradative compartment. Loading the cells with chloroquine did not affect this process. A monoclonal anti-CR1 Fab probe behaved in exactly the same manner, suggesting that the recycling of intact ligand-receptor complexes takes place. The possible physiological consequences of this finding are discussed.

Chloroquine↗
PubMed1988

Decline in rates of clearance of IgG-sensitized erythrocytes with increasing age.

Decreased immune functions have been suggested as a cause for the increased incidence of autoimmunity, malignancy, and infection in the elderly population. To assess the possible role of changes in macrophage function in the aging process we studied the Fc receptor-mediated clearance of IgG-coated erythrocytes in 56 healthy normal volunteers by following the removal of radiolabeled autologous erythrocytes. An age-related decrease in Fc-mediated clearance rates in both female and male subjects was found, which suggests a physiological decline of this macrophage function in older individuals.

Adult↗
PubMed1987

Complement component C3b binds directly to purified glycoprotein C of herpes simplex virus types 1 and 2.

Cells infected with herpes simplex virus type 1 (HSV-1), but not HSV-2, express on their surfaces a receptor for the complement component C3b. Receptor activity is markedly enhanced by treatment of the infected cells with neuraminidase. Employing a direct binding assay, consisting of purified HSV glycoproteins immobilized on nitrocellulose and iodinated C3b as a probe, we found that C3b binds directly to gC-1, as well as to gC-2, but not to gB or gD from either serotype. C3b binding was enhanced by treatment of gC-1 or gC-2 with neuraminidase. Endo F or endo H treatment of gC-1 had no effect on C3b binding. However, treatment of gC-2 with these endoglycosidases had a marked negative effect on C3b binding. These results suggest that N-linked oligosaccharides are involved in binding of C3b to gC-2, but not gC-1. Alternatively, removal of N-linked oligosaccharides from gC-2 might adversely affect polypeptide conformation. Glycoprotein C-2 also differs from gC-1 in its effects on the complement cascade. Whereas gC-1 accelerated the decay of the alternative pathway C3 convertase and impaired the efficiency of lysis by the components C5 through C9, gC-2 stabilized the active C3 convertase and had little effect on the late-acting components. The dissimilarity of gC-1 and gC-2 with regard to their effects on the complement cascade may have implications regarding the role of these glycoproteins in confronting the host immune response.

Animals↗
PubMed1987

Interactions of monomeric IgG bearing covalently bound C3b with polymorphonuclear leucocytes.

The interactions of monomeric IgG, monomeric C3b, dimeric C3b, and C3b covalently bound to IgG (C3b-IgG) with neutrophils were examined. C3b-IgG heterodimers exhibited an increased affinity of binding to neutrophils, relative to C3b, both at half normal ionic strength (five-fold enhancement) and at normal ionic strength (six-fold enhancement). Binding of monomeric IgG to neutrophils was barely detectable and did not permit direct measurements of affinity in our assay conditions. The increased affinity of C3b-IgG for neutrophils was the result of divalent interactions with CR1, the C3b receptor, and the Fc gamma receptor of the cells, as could be demonstrated by competition studies using unlabelled IgG in the medium or monoclonal antibodies against the neutrophil Fc gamma receptor. This double receptor-ligand interaction allowed C3b-IgG to bind to the cells not only at normal ionic strength, but also in the presence of a 315-fold excess of IgG (near plasma concentration)--conditions that would preclude the binding of C3b or IgG alone. Furthermore, this interaction leads to the internalization of the C3b-IgG complex by the cells. C3b-IgG is internalized with kinetics similar to those found using dimeric C3b, with resting cells demonstrating a slow initial phase, which is accelerated by phorbol ester activation in both cases. Uptake of the homodimeric C3b was greater at 15 min than that of heterodimeric C3b-IgG, but both were significantly greater than that of monomer C3b, which showed no net internalization. The physiological implications of these findings are discussed.

Complement C3b↗
PubMed1987

Phagocytosis of target particles bearing C3b-IgG covalent complexes by human monocytes and polymorphonuclear leucocytes.

Immunoglobulin G (IgG) provides an efficient acceptor site for nascent C3b, and complement activation on the surface of IgG-coated bacteria has been shown to generate significant numbers of C3b-IgG complexes. We have studied the relative efficiency of IgG alone, C3b-IgG complexes, and similar densities of IgG and C3b residues deposited independently, in mediating ingestion of sheep erythrocyte (E) targets by human phagocytes. Human 125I-C3b covalently bound to rabbit anti-Forssman IgG was generated as described elsewhere (Fries et al., 1985). E,EIgMC4b, or EIgMC4b3b (prepared with IgM antibody and purified complement components) were sensitized with radiolabelled anti-Forssman IgG or C3b-IgG heterodimers to generate targets bearing IgG alone, C3b-IgG covalent complexes, or C3b and IgG in equivalent numbers but not bound to each other. Phagocytosis by monocytes and polymorphonuclear leucocytes (PMN) of targets bearing C3b-IgG was markedly enhanced relative to those bearing IgG alone, especially at levels of less than 2000 opsonin residues/target cell. Uptake of C3b-IgG-bearing targets was also significantly more resistant to competitive inhibition by ambient monomeric IgG. Phagocytosis of EIgMC4b + C3b-IgG by monocytes was superior to the uptake of either EAC4b + IgG or EAC4b3b + IgG bearing equivalent amounts of C3b and IgG not in covalent complex (P less than 0.05, n = 10). Similar results were obtained with PMN. Thus, generation of C3b-IgG complexes in vivo may not only promote complement activation and enhance C3b deposition, but also produce a compound opsonic residue which is a more potent promoter of phagocytosis than an equal number of C3b and IgG residues randomly distributed relative to each other.

Animals↗
PubMed1986

Glycoprotein C of herpes simplex virus 1 is an inhibitor of the complement cascade.

Mammalian cells in culture express membrane receptors for C3b when infected with HSV-1. C3b binding is mediated by glycoprotein C (gC), a virus-specified membrane glycoprotein. In view of the inhibitory functions of other C3b-binding proteins, we studied the capacity of gC to modulate complement activation. Glycoprotein C was purified from HSV-1-infected cells by immunoaffinity chromatography. Glycoprotein C, but not a control viral glycoprotein, demonstrated dose-dependent acceleration of decay of C3bBb sites. In addition, gC produced a dose-dependent, time-independent depression of the overall hemolytic efficiency of C3bBb sites. Inhibition of C5b6-initiated reactive lysis of cells bearing C3b, but not cells bearing antibody alone, by gC suggests that the second effect represents interference with the C3b-C5/5b interaction. This hypothesis is supported by the failure of gC to inhibit reactive lysis when added after C5b67 insertion into target cells. Glycoprotein C does not accelerate C14b2a decay, nor does it impair classical pathway hemolytic efficiency when excess C5 is present. By limiting available C5/5b, some gC inhibition of C3b-C5/5b interactions can be unmasked in the classical pathway system. Glycoprotein C is devoid of factor I co-factor activity. HSV-1 gC is a modulator of complement activation, especially via the alternative pathway, and may represent a novel viral mechanism for evading host defense processes.

Animals↗
PubMed1986

Inherited deficiencies of complement and complement-related proteins.

The complement cascade and cell-surface proteins related to the complement system are critically important to host defense, immune complex catabolism, and possibly immunoregulation. Genetically determined deficiency states have been described for many complement proteins as well as for fluid phase and cell-borne inhibitors of the complement cascade and cellular complement receptors. Autoimmune diseases and enhanced susceptibility to infection are the dominant clinical manifestation of these deficiencies. Classical pathway deficiencies and low numeric expression of erythrocyte C3b receptors (CR1) are typically associated with autoimmune disorders, while alternative pathway, terminal component, and leukocyte iC3b receptor (CR3) defects predispose to pyogenic bacterial disease. This distinction is not absolute, however, and both classes of disease may appear in most of the reported deficiency states. Specific pharmacologic therapy exists for C1-inhibitor deficiency (hereditary angioedema). Management of other complement deficiencies currently involves sustained diagnostic suspicion and meticulous management of complicating diseases.

Anaphylatoxins↗
PubMed1985

IgG bearing covalently bound C3b has enhanced bactericidal activity for Escherichia coli 0111.

The mechanism was sought by which bactericidal IgG for E. coli 0111 (strain 12015) increases the bactericidal efficiency of C5b-9. IgG did not affect the distribution of C3 deposition on the O-Ag capsule and the outer membrane of 12015, suggesting that bactericidal IgG was not directing complement activation to different sites on the bacterial surface. However, one-fifth of the C3 that was deposited in the presence of IgG attached covalently to the antibody molecule. Covalent complexes between purified C3b and IgG were prepared in order to study the role of C3b-IgG in the bactericidal reaction. 8-10-fold less C3b-IgG than IgG was necessary to sensitize 12015 for serum killing. When aggregates were eliminated from the C3b-IgG and IgG preparations by sucrose density gradient ultracentrifugation, C3b-IgG remained three- to fourfold more effective than IgG on a molecule-for-molecule bound basis in mediating the serum bactericidal reaction. These results suggest that formation of C3b-IgG during the serum bactericidal reaction is critical for killing, and have important implications for the development of effective bactericidal vaccines.

Antibodies, Bacterial↗
PubMed1985

Factor I co-factor activity of CR1 overcomes the protective effect of IgG on covalently bound C3b residues.

We have shown previously that C3b resides in a protected site when it is covalently bound to IgG (C3b-IgG). Such C3b displays a reduced affinity for factor H, with consequent enhanced survival in the presence of factors H and I and increased capacity for promoting alternative pathway consumption of C3. Because erythrocyte CR1 may be a major co-factor for factor I-mediated inactivation of immune complex-borne C3b in blood, we have examined the effect of covalently bound IgG on the C3b-CR1 interaction. Binding of monomeric C3b and C3b-IgG to human erythrocyte CR1 demonstrates identical ionic strength dependence for both species. Identical numbers of binding sites with indistinguishable affinities are detected by both ligands. Cleavage of the alpha'-chain of C3b and the alpha'-heavy chain of C3b-IgG proceeds at the same rate when erythrocyte CR1 serves as co-factor for factor I. Unlike factor H, CR1 supports a second cleavage of fluid-phase iC3b alpha'1 chain (free or bound to IgG) that generates C3c and a 33,000 m.w. fragment, which bears antigenic markers characteristic of C3g. Inactivation of C3b and C3b-IgG by CR1 and factor I also occurs at physiologic ionic strength, but proceeds very slowly relative to rates attainable with sub-physiologic inputs of factor H. CR1 does not recognize IgG-bound C3b as being in a protected site but, because of low binding affinity at physiologic ionic strength, is probably highly dependent on multivalent ligand-receptor interactions to efficiently exert its co-factor functions. Thus, inactivation of C3b-IgG heterodimers or small immune complexes bearing limited numbers of C3b residues may remain largely factor H-dependent in vivo, with resultant enhanced C3b survival.

Antigen-Antibody Complex↗