Pharmacogenetics of hypersensitivity to abacavir: from PGx hypothesis to confirmation to clinical utility.

The hypersensitivity (HSR) to abacavir (ABC) pharmacogenetics (PGx) program represents the progression from an exploratory discovery to a validated biomarker. Within the program, two retrospective PGx studies were conducted to identify HIV-1 patients at increased risk for ABC HSR, a treatment-limiting and potentially life-threatening adverse event. A strong statistical association between the major histocompatibility complex allele, HLA-B*5701, and clinically diagnosed ABC HSR was identified but varied between racial populations. Subsequently, ABC skin patch testing was introduced as a research tool to supplement clinical case ascertainment. In a randomized, prospective study evaluating the clinical utility of HLA-B*5701 screening, avoidance of ABC in HLA-B*5701-positive patients significantly reduced clinically diagnosed ABC HSR and eliminated patch test-positive ABC HSR. Finally, a retrospective PGx study supports the generalizability of the association across races. Prospective HLA-B*5701 screening should greatly reduce the incidence of ABC HSR by identifying patients at high risk for ABC HSR before they are treated.

Specificity of three anti-complement factor 3 monoclonal antibodies.

Monoclonal antibodies which recognize specific C3 fragments may be used to distinguish C3 cleavage products bound to organisms. We defined the specificity of three commercially available monoclonal antibodies by Western immunoblot analysis, enzyme-linked immunosorbent assay, and a quantitative flow cytometric technique. Two monoclonal antibodies with specificity for (i) an erythrocyte-bound C3d epitope or (ii) an erythrocyte-bound C3c epitope retained their specificity in all assays. However, the third monoclonal antibody with selectivity for erythrocyte-bound C3bi failed to retain specificity for C3bi bound to non-erythrocyte surfaces in each of our assays; binding instead to all C3 fragments containing the C3g domain. We postulate that erythrocyte C3b-binding surface proteins may alter the availability of certain C3b epitopes and influence observed anti-C3 monoclonal antibody specificity. We conclude that the specificity of monoclonal antibodies for C3 fragments should be confirmed with assays which do not employ erythrocytes or other surfaces bearing C3 receptors. Also, our quantitative flow cytometric technique is a potentially valuable tool for the enumeration of particle-bound C3 fragments.

An enzyme-linked immunoassay (ELISA) for measurement of lactoferrin.

An enzyme-linked immunosorbent assay has been developed for quantitation of lactoferrin (LF) in body fluids. An indirect double-sandwich method was used which allows a sensitivity of 3 ng LF/ml in samples of polymorphonuclear cell lysates and serum. Mean LF content of serum was 0.307 +/- 0.066 micrograms/ml (n = 18). Mean LF content of polymorphonuclear cells was 4.90 +/- 1.48 micrograms/10(6) PMN. Concentrations of LF were similar in serum and in plasma of EDTA anticoagulated blood. Advantages of this method include its rapidity, and radioactivity is not required.

Bacterial antigens and neutrophil granule proteins in middle ear effusions.

Otitis media with effusion is a significant cause of hearing loss in young children. We hypothesized that persistent bacterial antigens in middle ear effusions (MEEs) might act as chronic inflammatory stimuli causing release of neutrophil proteins. Concentrations of neutrophil lactoferrin and a 37-kd cationic bactericidal protein (CAP 37) were measured in 47 MEEs collected from 27 children at the time of tympanostomy tube placement. Antigens of Streptococcus pneumoniae were detected by latex particle agglutination and those of Haemophilus influenzae by dot-blot assay. Bacterial antigens were detectable in 24 (51%) of MEEs: S pneumoniae in 10 (21%), H influenzae in 12 (26%), and both antigens in 2 (4%). Concentrations of lactoferrin and CAP 37 in H influenzae antigen-positive MEEs were significantly higher than in either S pneumoniae antigen-positive or antigen-negative MEEs. We conclude that H influenzae antigen causes a greater middle-ear inflammatory response, as judged by neutrophil products, than does S pneumoniae antigen.

Bacterial and polymorphonuclear leukocyte contribution to middle ear inflammation in chronic otitis media with effusion.

Bacteria can be cultured from approximately one third of chronic middle ear effusions, yet the contribution of these bacteria to the pathogenesis of chronic otitis media with effusion (OME) is not clear due to the absence of signs and symptoms of acute infection in most children with this disease. To explore the role of bacteria in chronic OME, lysozyme, lactoferrin, serum complement factors C3 and C5a, and polymorphonuclear leukocyte (PMNL) chemotaxin content was measured in 21 chronic middle ear effusion samples. Concentrations of lysozyme, lactoferrin, and chemotaxin were significantly higher in culture-positive than in sterile effusions. Lysozyme appeared to be contributed by both PMNL and non-PMNL sources in the middle ear space. These non-PMNL sources, presumably middle ear epithelial cells, accounted for 50% to 80% of the lysozyme variation in middle ear effusion. Although C3 and C5a were present in effusion, chemotaxin content correlated poorly with the C3 and C5a content, suggesting that chemotaxins were derived from bacterial peptides rather than from complement activation products. These results suggest that bacteria contribute to chronic middle ear inflammation with effusion. The eradication of bacteria from chronic middle ear effusion might disrupt the host responses which maintain chronic OME.

The intrinsic affinity constant (K) of anticapsular antibody to oligosaccharides of Haemophilus influenzae type b.

Antibody to the polyribosylribitol phosphate (PRP) capsular polysaccharide of Haemophilus influenzae type b is crucial to host defense. Affinities of antibody elicited by vaccination with PRP and PRP-diphtheria toxoid conjugate were determined using oligosaccharides (OS) from PRP. The affinities of antibody induced by vaccination with PRP to OS of three and four repeat units were similar but greater than the affinity to the two-unit OS. NaBH4 reduction of the three-unit OS did not alter the binding affinity, indicating that the reducing end of the OS did not participate in antibody binding. Over the range of OS concentrations tested, antibody affinity appeared to be homogeneous. Antibody concentration could be determined from binding experiments independently from affinity. Whole serum had 8- to 40-fold less antibody detected by binding analysis than by RIA, but the antibody concentration of an IgG fraction measured by the two methods agreed within a factor of two. We could not account for the discrepancy in concentrations found with whole serum by the presence of IgM or IgA antibody. The average affinity of antibodies of 10 adults vaccinated with PRP was similar to that of antibodies elicited in 14 adults vaccinated with a PRP-diphtheria toxoid conjugate (10.7 x 10(5) vs 7.6 x 10(5) liter/mol, respectively, p greater than 0.05). We conclude that the intrinsic affinity of antibody after vaccination with PRP is low and is not different from that of antibody elicited by PRP diphtheria toxoid conjugate.

Antibody to the outer membrane proteins is the dominant opsonic antibody in normal human serum against H. influenzae type b.

We hypothesized that the predominant opsonic antibody of normal serum is directed against outer membrane proteins (OMP). Sera from 10 normal adults were tested for their opsonic capacity against Haemophilus influenzae b (Eagan strain) by the luminol-enhanced neutrophil chemiluminescence elicited on incubation with serum-opsonized bacteria, and the ability to deposit C3 on the bacterial surface. Peak chemiluminescence correlated with the amount of C3 on the bacterial surface (r = 0.71, P less than 0.025) and this, in turn, correlated with the concentration of IgG directed against outer membrane proteins, (r = 0.75, P less than 0.01), but not with the concentration of anticapsular polysaccharide antibody. Two groups of sera were easily distinguished based on the chemiluminescence experiments: a high opsonic group (greater than 50,000 peak counts per second; c.p.s.) and a low opsonic group (less than 10,000 c.p.s.). The IgG fraction from the high opsonic sera could augment C3 deposition when added to a low opsonic serum, but could not after absorption of the anti-OMP antibody by affinity chromatography. We conclude that the predominant opsonin of normal serum is antibody to outer membrane proteins, a finding which could be significant for the development of future vaccines against H. influenzae b.

Opsonic activity of immunoglobulin prepared for intravenous use.

The opsonic activity of two immunoglobulin preparations modified for intravenous infusion was tested against Streptococcus pneumoniae types 3, 7F, and 14 and two strains of Staphylococcus aureus by polymorphonuclear leukocyte uptake of 3H-thymidine-labeled bacteria. Reduced and alkylated immunoglobulin (Chem-IgG) and immunoglobulin prepared by chromatography with diethylaminoethyl-Sephadex (DEAE-IgG) were evaluated with and without complement and compared with the opsonic activity of immune serum globulin and heated pooled human serum. Opsonic activity of DEAE-IgG was greater than that of Chem-IgG and equivalent to the activity of immune serum globulin and pooled human serum against S. aureus 502A and type 3 pneumococcus. Both intravenous immunoglobulins had lower opsonic activity than either pooled human serum or immune serum globulin against type 14 pneumococcus. There were no differences in antibody avidity for pneumococcal antigen among the immunoglobulins tested. All four opsonins had similar opsonic activity against the protein A-deficient S. aureus Wood 46. Modification of immunoglobulin for intravenous infusion by chemical alteration may adversely affect opsonic activity by changing the Fc portion of the antibody molecule.

Antibody affinity in infants after immunization with conjugated capsular polysaccharide from Haemophilus influenzae type b.

The affinities of IgG antibodies to Haemophilus influenzae b capsular polysaccharide (polyribosyl ribitol phosphate [PRP]) elicited 1 month after immunization of 47 infants 2-greater than 18 months of age with a PRP-outer membrane protein conjugate (PRP-OMP) were measured by ELISA. Thirty-four sera had affinities distributed normally about a logarithmic mean of 3.2 x 10(5) l/mol, but 13 samples had undetectable affinities (less than 10(4) l/mol). Median affinities of sera from children 2-6 (1.5 x 10(5) l/mol) and 7-11 months of age (1.6 x 10(5) l/mol) were significantly greater than the median affinities of sera from infants 12-18 (1.8 x 10(4) l/mol) or greater than 18 months of age (4.2 x 10(4) l/mol). Sera from children greater than 18 months of age vaccinated with PRP conjugated to diphtheria toxoid had a median affinity of 6.1 x 10(4) l/mol, equivalent to that of the same age group vaccinated with PRP-OMP. Children vaccinated with PRP conjugate vaccines may produce antibodies of very low affinity, a finding that may have significance for protection from invasive disease.

Correlation between antibody affinity and serum bactericidal activity in infants.

Nearly one-half of infants immunized with Haemophilus influenzae b capsular polysaccharide (polyribosylribitol phosphate; PRP)-protein conjugate produce low-affinity antibody. To test the hypothesis that antibody affinity is linked to biologic function, sera were obtained before and 1 month after immunization of 18-month-old infants with PRP-diphtheria toxoid conjugate vaccine. Correlation was attempted of anti-PRP affinity, concentrations of anti-PRP, and anti-outer membrane proteins and of immunoglobulin isotype with bactericidal activity. Nine subjects produced anti-PRP of low affinity (K less than 10(4) l/mol), and 11 had higher affinity antibodies (average K, 2.8 x 10(4) l/mol). By multiple regression analysis, antibody affinity was the only variable significantly related to the bactericidal activity of serum after immunization with the conjugate vaccine (r = .71; P = .04). Thus, serum anti-PRP from a substantial proportion of infants appeared functionally deficient in association with low-affinity antibody.

Complement component 3 binding to Haemophilus influenzae type b in the presence of anticapsular and anti-outer membrane antibodies.

Antibodies directed against the capsular polysaccharide (polyribosyl ribitol phosphate [PRP]) or the outer membrane proteins (OMP) of Haemophilus influenzae type b (Hib) promote bactericidal activity, complement 3 (C3) binding, and ingestion by phagocytic cells. To assess the relative contribution of anti-OMP to host defense against Hib, we compared the opsonic activities of anti-PRP and anti-OMP as reflected by the amounts of C3 bound to the bacterial surface. Immunoglobulin G (IgG) fractions containing either anti-PRP or anti-OMP were incubated with Hib in the presence of a C5-deficient complement source. C3, total IgG, and IgG subclasses bound to the bacteria were quantified by enzyme-linked immunosorbent assay. The maximum amount of C3 which could be bound to Hib was greater in the presence of anti-PRP than in the presence of anti-OMP. Also, except at low IgG concentrations, the rate of increase in bound C3 as a function of increasing IgG concentration was greater for anti-PRP than for anti-OMP. Hib-bound anti-OMP consisted primarily of IgG1 and IgG3, whereas bound anti-PRP was primarily IgG1 and IgG2. Thus, the potential for C3 binding to Hib is greater in the presence of anti-PRP than in the presence of anti-OMP, probably because of the larger number of binding sites available to the former. Nonetheless, OMP appear to provide important targets for opsonic antibody and would be logical components of a PRP-conjugate vaccine or may be efficacious as vaccines against nontypeable H. influenzae.

Human polymorphonuclear leukocytes of the bone marrow, circulation, and marginated pool: function and granule protein content.

Polymorphonuclear leukocytes (PMN) demonstrate altered function during acute infections and after administration of corticosteroids. We questioned whether or not such changes are due to population shifts from functionally different compartments of the granulocyte pool. Volunteers were given epinephrine to induce demargination or hydrocortisone (HC) to promote egress of PMN from the bone marrow. PMN obtained before and after drug administration were compared for adherence, chemotaxis, luminol-enhanced chemiluminescence, and total content and release of lactoferrin (LF), myeloperoxidase (MPO), and beta-glucuronidase (beta-glu). Epinephrine induced a significant neutrophilia of mature PMN (segmented neutrophils), but there were no changes in function or granule protein content. HC induced a significant neutrophilia with segmented neutrophils and immature PMN (bands). Circulating PMN obtained 4 hr after HC administration demonstrated less adherence, increased chemiluminescence, increased MPO release, and decreased MPO content. Band neutrophils, however, were more adherent than segmented PMN and showed a similar decrease in adherence following HC in vivo. Thus alteration of PMN adherence following intravenous corticosteroids is not due to an influx of immature neutrophils. On the other hand, it is possible that MPO content and release and capacity for oxidative metabolism change as PMN mature.

Outer membrane protein binding sites of complement component 3 during opsonization of Haemophilus influenzae.

Complement component 3 (C3) binding to Haemophilus influenzae type b (Hib) is an important step in host defense against invasive disease, but the details of this process remain poorly understood. We have shown that the P1 and P2 outer membrane proteins (OMPs) serve as binding sites for C3 on serum-opsonized Hib. Whole-cell lysates of opsonized Hib were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the resolved proteins were transferred to nitrocellulose. Immunoblot analysis with monoclonal antibodies (MAbs) to the 49-kDa P1 and 39-kDa P2 OMPs demonstrated high-molecular-weight bands that were not present when the bacteria were opsonized with heat-inactivated or methylamine-treated serum. Immunoblot analysis with MAbs to the 98- or 16-kDa (P6) OMPs did not reveal additional bands. An unencapsulated Hib mutant still lacked C3 bound to the 98-kDa or P6 OMP, indicating that the absence of C3 binding to these proteins was not the result of epitope masking by the capsule. Studies with MAbs to C3 fragments confirmed that the anti-P1- and anti-P2-reactive bands were C3 fragments bound to these OMPs. The molecular weights of proteins reactive to anti-OMP and anti-C3 antibodies indicated that multiple C3 fragments may be bound to P1 or that C3 may be bound to P2 multimers. Finally, the presence of other anti-C3-reactive proteins indicated that several other proteins serve as C3 targets during the opsonization of Hib.

Complement binding to Aspergillus conidia correlates with pathogenicity.

Complement has significant effects on the phagocytosis of Aspergillus organisms. We examined the amount and type of complement component C3 bound to the resting conidia of 29 isolates from nine Aspergillus species. The highly pathogenic species A. fumigatus and A. flavus bound fewer C3 molecules per unit of conidial surface area than did the less pathogenic species A. glaucus, A. nidulans, A. niger, A. ochraceus, A. terreus, A. versicolor, and A. wentii, as determined by quantitative flow cytometry. Immunoblot analysis of C3 fragments bound to conidia demonstrated that for all species most C3b was apparently converted to iC3b. For seven species, iC3b was clearly the major C3 product recognized by immunoblotting. However, A. niger and A. nidulans appear to promote further breakdown of opsonic C3 fragments to C3dg. We found significant variations in size and C3 binding among isolates within the same species. Intraspecies variation may contribute to seemingly discrepant results obtained in studies of Aspergillus phagocytosis.

Sequence and analysis of the rpoB gene of Mycobacterium smegmatis.

The rpoB gene encodes the beta subunit of the DNA-dependent RNA polymerase of bacteria. Mutations in defined areas result in resistance to rifampin. Mycobacterium smegmatis is naturally resistant to rifampin, but analysis of the rpoB gene revealed no identifiable rifampin resistance mutations. Another mechanism of resistance may be present.

Role of the Staphylococcus epidermidis slime layer in experimental tunnel tract infections.

An experimental animal model was used to assess the slime layer of Staphylococcus epidermidis as a pathogenic factor in tunnel tract infections. Mice were inoculated with high-slime-producing or non-slime-producing strains of S. epidermidis, either along the length of a subcutaneous catheter or in the area where a catheter had been placed and immediately removed (controls). Among the catheter-bearing mice, the phenotypically distinct staphylococci produced similar, high frequencies of abscess formation (72% [44 of 61] versus 81% [31 of 38]; P = 0.29). In controls, the non-slime-producing organisms were significantly more pathogenic (87% [40 of 46] versus 57% [25 of 44] abscess formation; P = 0.001). No consistent difference was detected between blood isolates obtained from patients with central venous catheter bacteremia and those from neonates with bacteremia in the absence of a prosthetic medical device. Quantitative culture of removed catheters showed greater adherence by the slime-producing isolates (P = 0.014). In this mouse model, slime production by S. epidermidis did not increase the risk of catheter tunnel tract infection, despite the greater catheter adherence of the slime-producing organisms. These findings suggest that traumatized tissue may be a sufficient condition for the development of S. epidermidis catheter-associated infections.

Functional capacities of clonal antibodies to Haemophilus influenzae type b polysaccharide.

Haemophilus influenzae type b (Hib) is an important pathogen for young children, and children can be protected with antibodies (Abs) to Hib polysaccharide (PS) capsule, a linear polymer of ribosyl ribitol phosphate. The structure of anti-Hib-PS Abs has been well characterized at the molecular level; about two-thirds of anti-Hib-PS Abs use a V kappa gene named A2, and the remaining anti-Hib-PS Abs use one of many other VL genes. In order to understand the structural basis for the variability in the function of these Abs, we prepared 18 clonally pure Abs from adults and studied their affinity, avidity, bactericidal potency in vitro, and ability to reduce bacteremia in newborn rats. Affinities and avidities were determined as the inverse of the concentrations of short (3 repeating units) and long (20 repeating units) ligands which could bind 50% of anti-Hib-PS Ab in solution, respectively. No significant correlations between the protection of newborn rats and affinity (r = 0.02) or avidity (r = 0.16) were observed. The amount of Ab required to kill 50% of bacteria in vitro decreased with avidity (r = -0.32), as expected. However, Abs with high affinity were unexpectedly found to have less bactericidal activity (r = 0.38). This suggests that avidity may be a better predictor of Ab function than affinity. Affinity and avidity results were negatively correlated (r = 0.76, P = 0.0022), and Abs that had A2 V kappa gene products had higher avidity (P < 0.05) and lower affinity (P = 0.06) than Abs that had other VL genes. A possible explanation of these observations is that the epitope for Abs with the A2 gene is within the Hib-PS chain itself, whereas the epitope for Abs with a non-A2 gene is the terminus of Hib-PS.