Opsonization of Actinobacillus actinomycetemcomitans by immunoglobulin G antibodies to the O polysaccharide of lipopolysaccharide.

Sera of localized juvenile periodontitis (LJP) patients colonized by Actinobacillus actinomycetemcomitans serotype b often contain markedly elevated levels of immunoglobulin G (IgG) antibodies to serospecific determinants in the O polysaccharide of lipopolysaccharide (LPS), as well as to outer membrane proteins of this species. IgG antibodies in LJP sera are known to opsonize A. actinomycetemcomitans for subsequent phagocytosis and killing by human neutrophils. The objective of this study was to determine whether outer membrane proteins or serospecific determinants in LPS are the primary target for opsonic IgG antibodies in LJP sera. An A. actinomycetemcomitans serotype b O-polysaccharide affinity column was constructed and subsequently used to purify LPS-specific IgG antibodies from LJP serum. The affinity-purified anti-LPS IgG antibodies were enriched in content of IgG2 (66.2%, compared with 37.0% in the total IgG fraction) and were immunospecific for A. actinomycetemcomitans serotype b LPS. In an opsonophagocytic assay using neutrophils from donors who were homozygous for the H131 allotype of Fcy receptor IIa (CD32), it was found that LPS-specific IgG antibodies exhibited substantially greater opsonic activity toward A. actinomycetemcomitans serotype b than an LJP IgG fraction that was depleted of LPS-reactive antibodies but contained antibodies against outer membrane proteins of this species. The results of this study indicate that serospecific determinants in the O polysaccharide of A. actinomycetemcomitans serotype b are a principal target for opsonic antibodies in sera of LJP subjects.

Immunoglobulin G subclass response to major outer membrane proteins of nontypable Haemophilus influenzae in children with acute otitis media.

Children with acute otitis media as the result of nontypable Haemophilus influenzae often develop serum bactericidal and/or opsonic IgG antibodies to this organism during convalescence. Outer membrane proteins appear to be the principal targets for such antibodies. In this study we characterized the IgG subclass responses to major outer membrane proteins of nontypable H. influenzae in otitis-prone children in whom this organism had colonized. Three of the major outer membrane proteins (P2, P5, and P6) were isolated from the homologous nontypable H. influenzae strain recovered from the middle ear at the time of acute infection. Sera were obtained during the acute phase and at 1 and 6 months thereafter. The outer membrane proteins, which were isolated by preparative sodium dodecylsulfate-polyacrylamide gel electrophoresis, were used as test antigens in a quantitative IgG subclass enzyme immunoassay. The results of this analysis indicate that the temporal characteristics and distribution of IgG subclass antibodies were found to differ for each of the outer membrane proteins. Moreover, substantial variation between patients was observed with respect to both temporal characteristics and subclass distribution of the IgG response to the three outer membrane proteins. Significantly, sera from two of three otitis-prone subjects contained detectable levels of IgG antibody to the conserved P6 outer membrane protein at the time of acute infection, with serum from one subject also containing detectable levels of IgG3 antibody to this same protein. Nevertheless, the organism persisted in the middle ears of these patients. The results of this study indicate that otitis-prone children manifest a highly variable IgG subclass response to both conserved (P6) and variable (P2) outer membrane proteins of nontypable H. influenzae. Further study is required to ascertain whether these IgG subclass antibodies are biologically efficacious and whether otitis-prone children possess the immunologic maturity to respond to nontypable H. influenzae outer membrane protein-based vaccines in a predictable manner.

Immunoglobulin G2 antibodies promote neutrophil killing of Actinobacillus actinomycetemcomitans.

Sera from patients with localized juvenile periodontitis (LJP) often contain markedly elevated levels of immunoglobulin G2 (IgG2) antibodies reactive to cell envelope constituents of Actinobacillus actinomycetemcomitans. The objective of this study was to determine if these IgG2 antibodies are capable of supporting phagocytosis and killing of A. actinomycetemcomitans by human neutrophils. Polyclonal IgG2 antibodies were prepared from high-titer LJP serum by affinity chromatography, yielding a preparation which was > 99% subclass restricted and retained immunoreactivity to A. actinomycetemcomitans antigens. Affinity-purified IgG2 antibodies were evaluated by an in vitro opsonophagocytic assay that employed neutrophils obtained from donors who were homozygous for the H131 allotype of Fc gamma receptor type IIa (CD32), which efficiently binds human IgG2 antibodies. Affinity-purified IgG2 antibodies from LJP serum but not from sera of periodontally healthy individuals promoted phagocytosis and killing of A. actinomycetemcomitans. The expression of IgG2-dependent opsonic activity required the presence of complement. Incubation of A. actinomycetemcomitans with neutrophils in the presence of an optimal concentration of LJP IgG2 (50 micrograms/ml) and 5% hypogammaglobulinemic serum (as a complement source) resulted in a > 1 log10 reduction in bacterial viability within 30 min. The opsonic activity of IgG2 antibodies was found to be comparable to that observed with affinity-purified IgG1 antibodies. Moreover, IgG1 antibodies interacted synergistically with IgG2 antibodies in promoting opsonophagocytosis of A. actinomycetemcomitans. The results of this study indicate that LJP serum contains IgG2 antibodies which, when employed in conjunction with neutrophils that express Fc gamma receptors capable of recognizing this subclass, are opsonic for A. actinomycetemcomitans.

Capnocytophaga species: increased resistance of clinical isolates to serum bactericidal action.

Capnocytophaga, a newly recognized genus of capnophilic gram-negative bacilli, is part of the normal oral flora. The capacity of Capnocytophaga to cause sepsis and local infections in both immunocompromised and nonimmunocompromised hosts has been documented. Given the recognition that serum resistance may contribute to the virulence of some gram-negative bacteria, we attempted to define the serum sensitivity of clinical isolates of Capnocytophaga from blood and other sites of infection. Whereas nine of nine isolates from human subgingival plaque showed greater than 95% loss of viability under standardized assay conditions, nonoral isolates exhibited variable serum sensitivity. Six of six isolates from blood showed considerable serum resistance (mean survival, 59.7% +/- 38.3%; range, 14.4%-113.3%). Comparison of the electrophoretic mobilities of lipopolysaccharides (LPSs) from sensitive and resistant strains revealed reduced LPS heterogeneity and lower apparent molecular weight among serum-resistant strains. Thus, serum resistance, possibly influenced by LPS structure, may be an important factor contributing to the pathogenic potential of Capnocytophaga spp.

Human oviductal fluid proteins. II. Preparation of an antiserum to a human oviductal fluid protein: existence of autoantibodies against it in some sera.

Rabbit antisera elicited by injection of human oviductal fluid (HOF) and rendered specific by absorption with human kidney and human serum revealed the presence of two possibly HOF-specific antigen(s). One antigen was detected by immunodiffusion in 14 of the 14 fluids tested with one of these antisera. HOF-specific antigen was shown to be a beta-globulin by immunoelectrophoresis. A second antigen was seen in two samples. That autoantibodies might exist directed against HOF-specific antigen in the donor's serum was suggested by immunodiffusion experiments in which 4 of the 13 sera tested gave lines of precipitation when reacted against the corresponding oviductal fluid. In one instance, this line was the same as one of the two lines seen in tests using rabbit antiserum.

Affinity diffusion. I. Method for measuring dissociation constants of precipitating antibodies.

Dissociation constant (Kd) of antigen-antibody reactions can be obtained from the rates (measured by the progression of the precipitate vs. time) with which antigens diffuse into antibody-containing gels, as a function of antibody-concentration. A bovine serum albumin vs. rabbit anti-bovine serum albumin system was studied with whole antiserum and with its purified IgG fraction. A value was found for Kd of approximately 1.0 x 10-5 moles per liter. It is note- worthy that in monodimensional single diffusion gel precipitation systems of this type, the rate of progression of the precipitate front is significantly faster than the molecular diffusion coefficient of the antigen.

Affinity diffusion II. Comparison between thermodynamic data obtained by affinity diffusion and precipitation in tubes.

Association constants (Ka) of the precipitating system bovine serum albumin (BSA) goat anti-BSA were obtained at different temperatures via affinity diffusion (taking l/Kd - Ka) as well as via precipitation in tubes at optimal ratios. With affinity diffusion values of Ka of 0.6 to 1.1 x 10(5) l/M were found, whilst with precipitation in tubes Ka was from 3.3 to 11.2 X 10(7) l/M, using the same BSA and anti-BSA preparations. Via affinity diffusion binding energies delta F of approximately -6 to -7 kcal/M were found, with values of delta H close to zero, and a delta S of +23 entropy units. With precipitation in tubes these values were delta F -10.2 to -10.7 kcal/M, delta H -4.6 to -7.6 kcal/M and delta S +10 to +20 entropy units. The differences found with the two different methods must be ascribed to the fact that with affinity diffusion of precipitating antigen-antibody systems one just measures the interaction between the precipitating components with the highest dissociation constants, whilst with precipitation in tubes one measures the total energy of association of the system. With affinity diffusion and with precipitation in tubes, the same degree of positive entropy is observed. The system measured with affinity diffusion is approximately isothermic, whilst the total system, measured by precipitation in tubes, is strongly exothermic. Affinity diffusion still takes place at pH 9.5, at which pH no precipitation in the liquid phase takes place at optimal ratio; one may conclude from this that affinity diffusion mainly involves van der Waals interactions, as electrostatic bonding between BSA and anti-BSA is virtually abolished at that pH. This agrees well with the observation that the affinity diffusion reaction is isothermic.

Two methods for the removal of erythrocytes from buffy coats for the production of human leukocyte interferon.

Two different methods of removing erythrocytes from buffy coats, with the ultimate aim of inducing the white blood cells for the production of leukocyte interferon, i.e., erythrocyte lysis with ammonium chloride, and erythrocyte agglomeration with hydroxyethyl starch, are compared. After two treatments with 0.83% NH4Cl all erythrocytes are lysed and most granulocytes are damaged, although the lymphocytes appear mostly unaffected (as judged by microphotography of Wright-stained smears). Treatment with 3% hydroxyethyl starch causes most erythrocytes to agglomerate and sediment to the bottom of a vessel in less than one hour; the leukocytes (lymphocytes as well as granulocytes), which remain in suspension, are morphologically intact. NH4Cl-treated leukocytes yield, on an average, 3.6 times less interferon than hydroxyethyl starch-treated leukocytes. Leukocyte viability, as judged by trypan blue exclusion does not appear much influenced by either NH4Cl or hydroxyethyl starch treatment. The advantage of hydroxyethyl starch apparently lies in the fact that its use leaves the granulocytes intact and thus obviates the undue release of proteolytic enzymes into the culture medium, with their concomitant deleterious effect on interferon production.

Preparative electrophoresis of human lymphocytes. II. Use of non-immunoglobulin bearing lymphocytes obtained by electrophoretic levitation for the elicitation of a rabbit anti-human "T-cell" serum.

By intradermal injection (in Freund's complete adjuvant) of rabbits with only a few million human T-lymphocytes, obtained by preparative electrophoresis, an anti-human "T-cell" serum can be obtained. Although the antiserum is best used in a fairly concentrated form, its anti-human "T-cell" specificity (by immunofluorescence and E-rosetting) appears excellent, without the need to resort to absorption with various unwanted antigens. There may however, also be other specificities in the antiserum, as it also reacts with human granulocytes.

Preparative cell electrophoresis with D2O as a stabilizing agent.

The method of ascending preparative cell electrophoresis in a nonstabilized vertical column can be greatly improved by layering the cells on a starting cushion of buffer prepared in D2O and by stabilizing the liquid column above it with a D2O gradient. D2O/H2O mixtures have no apparent biochemical effects on the cells, and their physiochemical effects are short-lived and reversible. It was possible, by this method, to separate a mixture of glutaraldehyde-fixed erythrocytes of three species (rabbit, horse and chicken) into three distinct zones after 15 minutes of electrophoresis. It was also possible to obtain an enriched human lymphocyte fraction containing 96% T-cells, after 1 hour of electrophoresis.

Changes in the serum alpha glycoprotein distribution in trauma patients.

Serum electropherograms of trauma patients, when stained for glycoproteins, show striking changes in the alpha glycoproteins of these patients which revert to normal if and when they recover. Thus monitoring of the glycoprotidograms of trauma patients (contrary to ordinary protidograms) is likely to afford important information on the course of these patients' recovery. Of two glycoproteins that have been more closely studied, one, alpha1A acid glycoprotein, is a phagocytosis inhibitor; it is increased in the sera of trauma patients. The other, alpha2HS glycoprotein, is a phagocytosis promotor (or opsonin); it is decreased in the sera of trauma patients. Both the increase of the first and the decrease of the second glycoprotein may thus contribute to the known increased proneness to bacterial infection among these patients.