Alterations of the glycocalyx of Fc receptor-bearing cell lines during Fc receptor-ligand interactions

The binding of IgG-coated erythrocytes to Fc receptors on both a lymphoblastoid and a macrophage-like cell line resulted in a decrease in thickness of the polyanionic, extracellular glycocalyx (cell coat) as determined by electron microscopic histochemistry. This decrease showed no correlation with ligand-binding sites and was considered to be a generalized extramembrane effect. Pretreatment of the cells with trypsin or neuraminidase produced decreases in thickness similar to those observed following ligand binding. The results suggest a possible role for enzymatic cleavage of extracellular constituents by morphologically and functionally different cell types and may represent an event common to cell-surface recognition.     

Fc receptors and their interaction with complement in autoimmunity

Genetic studies in mice indicate a crucial role for Fc receptors (FcR) in antibody-mediated autoimmune diseases. Like other immune regulatory receptor pairs, the FcR system is constituted by activating and inhibitory receptors that bind the same ligand, the Fc portion of Ig. Analyses of animal models have shown that the inhibitory Fc receptor, FcgammaRIIB can suppress antibody-mediated autoimmunity, whereas activating-type FcR, such as FcgammaRIII promote disease development. This review summarizes recent advances of FcR, as obtained from gene deletion studies in mice, and highlights the importance of factors that interact with FcR in autoimmunity. There is emerging evidence for an indispensable role of the complement component C5a in the regulation of FcR and the sensing of FcR-dependent effector cell responses. On the other hand, FcR might be alternatives to serum complement in the generation of C5a at sites of inflammation. Thus, FcR and complement interact with each other at the level of C5a by linking regulatory events with effector cell activities in autoimmunity. This connecting pathway is now proposed to be a promising new therapeutic target for the treatment of inflammation and autoimmune disease in both mice and humans.     

Optimal oxygen tension conditions for viability and functioning of precision-cut liver slices.

Optimal oxygenation of culture media is important for the successful use of liver slices as an in vitro tool for studying liver function. For this reason the influence of 20, 40, 70 and 95% O2 concentration on the viability and metabolism of liver slices was investigated. The slices were incubated in the roller system at 37 degrees C under continuous gassing for 2, 24 and 48 hrs. Protein, DNA and potassium contents were maintained or even increased over time without influence by O2 concentrations. The albumin secretion of slices incubated at 40-95% O2 did not differ, but was much lower at 20% O2. A slight non-significant decrease in albumin secretion after 24 hrs of cultivation could be observed, whereas a much steeper decline was found in all groups after 48 hrs. Cytochrome P450 (CYP)-dependent 7-ethoxycoumarin O-deethylation (ECOD) did not differ between the various O2 concentrations, but declined from 2 to 48 hrs of incubation. It can be concluded that O2 concentration of 20% is not sufficient to maintain all cell functions of incubated rat liver slices, wheras 40, 70 and 95% are useful O2 concentrations to retain all parameters investigated.     

Generation of canine-human Fc IgE chimeric antibodies for the determination of the canine IgE domain of interaction with Fc epsilon RI alpha

Identification of the domain(s) of canine IgE that interact with Fc epsilon RI alpha may lead to novel therapeutic intervention strategies that inhibit the ability of canine IgE to engage Fc epsilon RI alpha. A panel of canine-human Fc IgE chimeric antibodies was constructed to investigate this interaction by replacing canine IgE-Fc domains with the corresponding human IgE-Fc domains since human IgE-Fc does not recognize canine Fc epsilon RI alpha. beta-Hexosaminidase release assays were performed to assess the ability of the chimeric antibodies to bind to and sensitize a novel RBL cell line transfected with canine Fc epsilon RI alpha for antigen induced mediator release. Replacing canine C epsilon2 with human C epsilon2 resulted in similar levels of release as those elicited by canine Fc IgE from RBL-2H3 cells transfected with either canine Fc epsilon RI alpha or human Fc epsilon RI alpha. Substitution of canine C epsilon4 with human C epsilon4 resulted in approximately 10% lower levels of release compared to cells sensitized with canine Fc IgE. Receptor binding by flow cytometry and cell activation could not be detected when transfected RBL cells were incubated with chimeric constructs where canine C epsilon2 and C epsilon4 were substituted with human C epsilon2 and C epsilon4. However, when this construct was incubated with cognate antigen prior to cell challenge mediator release was observed, albeit at a 20% lower level, indicating that while canine C epsilon3 is the only domain essential for binding to canine or human Fc epsilon RI alpha, species specific residues in canine Cepsilon2 and C epsilon4 inhibit dissociation of the ligand from the receptor.     

A microassay for studies of the interaction between Fc receptors and low affinity ligands

A microassay using 10 microliter of a conjugate of rat IgG and Sepharose was developed for the study of the interaction between the solubilized receptors for IgE of rat basophilic leukemia (RBL) cells, designated H and R, and a variety of low affinity immunoglobulins. Rat IgG2a was used as the model of a low affinity ligand by serving as an inhibitor of the interaction between IgG-Sepharose and the receptors for IgE. Ligand concentrations 2.24 X 10(-7) to 5.37 X 10(-5) M were used along with detergent extracts of 2 X 10(5) RBL cells.     

Identification of the site on IgG Fc for interaction with streptococci of groups A, C and G.

The interaction between living groups A, C and G streptococci and IgG Fc was studied using human IgG, IgG Fc and IgG Fc-intermediate (Fci) fragments, chemically modified human IgG and fragment D of staphylococcal protein A (SPA). Diethylpyrocarbonate modification of His or N-acetylimidazole modification of Tyr of human IgG resulted in the loss of its capacity to inhibit the binding of radiolabelled human IgG Fc to the group A streptococci types M1 and M55, and to the group C strain SC-1, indicating that the amino acids His and Tyr are involved in the binding. Lys seems not to participate in the binding of IgG to these bacteria, however, since reductive methylation of Lys did not reduce its inhibitory capacity. Fragment D of SPA also inhibited the binding of radiolabelled human IgG Fc to strains M1, M55 and SC-1. We have previously shown that these bacteria do not bind to IgG fragments consisting of only the C gamma 2 or C gamma 3 domains. On the basis of these results, and the known relative positions in space of the His and Tyr residues on IgG Fc, it is speculated whether streptococci with IgG Fc receptors, like SPA and rheumatoid factors, interact with IgG in the interface between the C gamma 2 and C gamma 3 domains and involve His 435 and one or more of Tyr 436, His 433 and His 310. The similarities in binding sites on IgG for RFs and these bacterial Fc binding proteins suggest structural similarities between them that may be relevant to the production of rheumatoid factors in rheumatoid arthritis.     

Fc epsilon receptors.

Advances in our understanding of the molecular structure of Fc receptors have been made at a rapid pace. Details of how Fc receptors are involved in cell triggering, e.g. allergic mediator release from mast cells, and IgE synthesis are also continuing to be elucidated, although much work is still required. Recent highlights of investigations of mast-cell and lymphocyte IgE Fc receptors will be outlined.     

T-cell hybridoma co-expressing Fc receptors for different isotypes. I. Reciprocal regulation of Fc alpha R and Fc gamma R expression by IgA and interferon.

To clarify the co-expression phenomenon of T-cell Fc receptors (FcR) specific for different isotypes on the clonal level, a murine hybridoma clone T2D4 was studied. T2D4 cells originally reported to bear FcR for IgG (Fc gamma R) and to release a Fc gamma R-related T-cell factor binding to IgG (immunoglobulin binding factor; IBF) proved to have also the receptor for IgA. The binding of IgA was detected by rosette formation with trinitrophenylated ox red blood cells (TNP-ORBC) after preincubation of T2D4 cells with MOPC 315 IgA having anti-TNP activity, or directly with TNP-ORBC sensitized with MOPC 315 IgA. While the binding of MOPC 315 IgA was competed for by IgA but not by IgG2A nor IgG2B, IgA failed to inhibit the rosette formation of the cells with ORBC sensitized with rabbit IgG antibody (EA ox gamma), proving that T2D4 cells express FcR specific for IgA (Fc alpha R) in addition to Fc gamma R. Co-expression of both receptors on the same cell surface was demonstrated by a double rosette technique using TNP-quail red blood cells (TNP-QRBC) and EAox gamma. Fc alpha R activity of the cells was completely abrogated by 15 min. incubation with 0.1 mg/ml trypsin, whereas Fc gamma R was resistant even to 1 mg/ml trypsin. The expression of Fc alpha R was augmented (up-regulation) by IgA at the concentration above 300 micrograms/ml and inhibited (down-regulation) by 1000 u./ml of murine beta-interferon (beta-IFN). Conversely, the expression of Fc gamma R was down-regulated by IgA and up-regulated by alpha-IFN. Thus, Fc gamma R and Fc alpha R are co-expressed and reciprocally regulated on these cell lines. The possible co-production of IBF and the Fc alpha R-related binding factor specific for IgA is discussed.     

Optimal survival of Helicobacter pylori under various transport conditions.

The ability of clinical isolates and type strains of Helicobacter pylori to survive in Stuart transport medium, isotonic saline solution, and urea-containing isotonic saline was evaluated. The influences of temperature (4, 10, 15, 20, and 30 degrees C) and holding time (6 to 48 h) and the effect of exposure to air on survival were also studied. The recovery rate of H. pylori was highest from Stuart transport medium in comparison with the recoveries from the other transport media tested. We found that at holding temperatures above 15 degrees C the organisms became noncultivable within 6 h or less, while they survived for 2 days or longer at 10 degrees C. The presence of urea at a concentration of 2% (wt/vol) in isotonic saline resulted in the loss of viability of the organisms tested.     

Optimal conditions for elution of hepatitis B antigen after absorption onto colloidal silica.

Hepatitis B surface antigen (HBSAg) adsorbed from sera onto colloidal silica could be completely eluted through the use of 0.25% sodium deoxycholate in 0.01 M borax, pH 9.3, at 56 degrees C. The HBSAg recovered in the eluate represented 100% of that present in the original serum, and it was contaminated by only trace amounts of serum proteins (in decreasing amounts: beta-lipoprotein, immunoglobulin G, albumin). This preliminary step greatly facilitates purification of large amounts of HBSAg and provides small volumes of highly concentrated material for subsequent purification by density gradient centrifugation.     

Optimal conditions for demonstrating the antimitotic action of interferon

The effect of human leukocyte interferon (HLI) on three human cell cultures of different origin was studied. HLI inhibited the mitotic activity (MA) of human ovary carcinoma cell culture and to a lower extent that of a diploid cell culture. At the same time HLI preparation exerted a nonspecific stimulating effect on MA of the primary human embryo cell culture. The optimal conditions for the determination of the antimitotic effect of interferon on cells of tumor origin include: a seed dose of 5 X 10(4) cells/ml, addition of interferon to the culture 24 hours after cell seeding; the maximum effect of interferon is observed at 24 hours after addition to the culture.     

Optimal conditions for alumina coating formation on the MA956 superalloy for prosthetic bearing applications.

An experimental study of the oxidation treatment at high temperature of the ODS MA956 superalloy was conducted in an attempt to achieve a protective alumina scale for biomedical applications. A quadratic response-surface model was developed in order to study the effects of treatment time and temperature (in the range of 1000 degrees C to 1250 degrees C) on scale thickness. The obtained model adequately represents the experimental response and shows that the thickness gradients of the layer increase with the temperature for each exposure time and decrease steadily to zero as the treatment time increases. The microstructural characterization reveals that the alumina scale formed at or above 1000 degrees C consists of an alpha-alumina phase. Treatments at temperatures above 1150 degrees C give rise to an alumina scale with some defect probability. An increase in the temperature up to 1200 degrees C gives rise to the appearance of some blistering of the superficial scale. An oxidation treatment of 100 h at 1100 degrees C was found to be the best for guaranteeing the formation of a defect-free, compact, adherent, and continuous alpha-alumina scale thick enough to support satisfactory wear and biological conditions.     

Optimal conditions for recovery of the human immunodeficiency virus from peripheral blood mononuclear cells.

Optimal conditions for demonstrating the presence of infectious human immunodeficiency virus in peripheral blood mononuclear cells (PMCs) from seropositive individuals involved cocultivation of infected cells with phytohemagglutinin-stimulated PMCs from seronegative donors in the presence of 2 micrograms of Polybrene per ml. The size of the culture vessel also influenced the results; smaller numbers of infected cells were detected under conditions of increased cell density. In addition, an increased normal donor/patient PMC ratio was helpful. The cocultivation approach permitted identification of human immunodeficiency virus in over 90% of seropositive individuals with different clinical conditions. Moreover, reconstruction experiments indicated that this method allows detection of one productively infected CD4+ cell in a population of over 10(6) PMCs.     

Aspects of immunoglobulin G structure relevant to its interaction with Fc receptors

One unambiguous message that comes from the data obtained relates to the central role played by the hinge region, and its constituent disulfide bonds, in the functioning of the IgG molecule. Segmental flexibility allows variation in the distance between the antibody combining sites, located at the distal ends of the Fab regions, and serves the important function of allowing bivalent attachment of antibodies to antigenic determinants even if the latter form a fixed array, for example, on a cell surface (monogomous bivalency). Too much flexibility, however, seen as a consequence of cleaving the hinge-region disulfides appears to be inimicable to the expression of effector functions. The hinge region and its disulfides control the degree of flexibility, thus allowing the IgG molecule to perform its varied functions.     

Linear conditions in chain systems with neighbour interaction. II. Application of structural conditions: Atactic polypropylene

It is shown here how the theory proposed in the preceding paper may be applied to the conformational analysis of atactic polymers as well as of oligomers in solution. The calculation of the free mean-square lengths of polypropylenes with various types and degrees of tacticity is discussed in detail. In particular, the chain distributions of d and I units, coresponding to the models taken into consideration, are closely represented by the MARKOFFian law. The agreement between our results and those obtained by FLORY et al. through Monte-Carlo calculations is substantially good. The reasons why the configurational defects in nearly isotactic poly-α-olefins should be more closely represented by the (… dddlddd …) sequences than by the (… dddlll …) ones proposed by FLORY, avidenced.     

Mapping and comparison of the interaction sites on the Fc region of IgG responsible for triggering antibody dependent cellular cytotoxicity (ADCC) through different types of human Fc gamma receptor.

In the present study 3-iodo-4-hydroxy-5-nitrophenacetyl (NIP)-specific antibodies were compared for induction of antibody dependent lysis of NIP-derivatised red blood cells effected by pre-stimulated U937 or HL-60 cells and by K cells. The chimaeric antibodies have heavy chains corresponding to human IgG subclasses 1-4, and include site-directed mutants of IgG3 as well as the aglycosylated form of IgG3; a mouse IgG2b antibody and a site-directed mutant IgG2b were also examined. rIFN stimulated U937 or HL-60 cells express increased levels of Fc gamma R1 compared to unstimulated cells; PMA stimulated HL-60 and U937 cells express an increased level of Fc gamma R11 compared to unstimulated cells; K cells express Fc gamma R111. Using these effector cell populations and the target cells mentioned above, we have compared anti-NIP antibodies with different heavy chain constant domains for their ability to induce ADCC through human Fc gamma R1, Fc gamma R11 and Fc gamma R111. The results suggest that all three human Fc gamma receptors appear to recognise a binding site on IgG within the lower hinge (residues 234-237) and trigger ADCC via this site, but that each receptor sees this common site in a different way. The possibility that other amino acid residues also participate in the binding/triggering site(s) cannot be excluded.     

Genetic diversity in human Fc receptor II for immunoglobulin G: Fc gamma receptor IIA ligand-binding polymorphism.

Fc gamma receptors, and in particular genetic variation in these receptors, are important in disorders of hose defense, immunohematologic disease, and systemic autoimmune diseases. We investigated the His-Arg (CAT/CGT) polymorphism at codon 131 of the Fc gamma receptor IIA gene, which influences ligand binding by the receptor. Previously, individuals had been classified phenotypically on the basis of differential binding of murine immunoglobulin G1, but the Fc gamma receptor IIA genotype distribution has not been reported. We used selective PCR-based sequence analysis of genomic DNA to determine the distribution in healthy individuals. For African-Americans, the genotype distribution was determined to be A/A (14%), A/G (60%), and G/G (26%); for Caucasian Americans, the distribution was A/A (30%), A/G (51%), and G/G (19%). These data correlate well with phenotypic data. We implemented a nonradioactive single-stranded conformational polymorphism analysis to rapidly identify all three genotypes. The PCR-single-stranded conformational polymorphism analysis method will facilitate studies of the genotype distribution in individuals with disorders of immune function.