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Characterization of the human immune response to a polysaccharide vaccine from Pseudomonas aeruginosa.

Sera from humans vaccinated with a high-molecular-weight polysaccharide vaccine to Pseudomonas aeruginosa immunotype 1 (IT-1) were analyzed for duration of the immune response, specificity for the IT-1 determinant, and by assessing the immunoglobulin classes elicited. The ability of purified IgG, IgM, and IgA to interact with peripheral blood leukocytes, as well as purified polymorphonuclear neutrophils or mononuclear cells, was also examined in an opsonophagocytosis assay. Levels of antibody to IT-1 remained significantly (P less than 0.001) elevated 21 months after immunization. Responses were generally specific to the IT-1 serotype determinant. Some vaccinees also responded to immunotype 2 and immunotype 5 determinants. IgG, IgM, and IgA serum antibodies were all elicited by vaccination. IgG and IgA were effective opsonins for P aeruginosa. IgM-mediated opsonophagocytosis required complement. Serum IgA was highly effective in conjunction with mononuclear cells in opsonophagocytosis of P aeruginosa, suggesting that these immune components may be capable of protecting neutropenic hosts.

Adult↗

The contribution of human neutrophils and serum to host defense against Legionella micdadei.

The interaction of Legionella micdadei with human polymorphonuclear neutrophils (PMNs) and serum was investigated to determine their roles in host defense against this organism. Serum and PMNs from normal donors having no antibody to L micadei were used. PMNs phagocytized once-agar-passaged (74.6% +/- 6.5%) and twice-agar-passaged (87.3% +/- 1.0%) L micdadei less (P less than 0.05) than L micdadei passaged multiple times on agar (97.5% +/- 1.0%) or Staphylococcus aureus (98.3% +/- 0.5%). Under the same conditions, no phagocytosis of Legionella pneumophila occurred. Use of heat-inactivated serum abolished phagocytosis. PMN killing of once-agar-passaged (1.5% +/- 1.5%) and twice-agar-passaged (0) L micdadei was less (P less than 0.001) than that of L micdadei passaged multiple times on agar (88.6% +/- 4.0%) or S aureus (96.8% +/- 0.5%). L micdadei was not killed by fresh serum, although, in contrast to L pneumophila, it was opsonized by C3. Thus virulent L micdadei is phagocytized, but not killed, by human PMNs, with complement being the major opsonin.

Blood Bactericidal Activity↗

Quantitative variability in requirements for opsonization of strains within the Bacteroides fragilis group.

Requirements for opsonization of various strains within the species Bacteroides fragilis, Bacteroides thetaiotaomicron, Bacteroides vulgatus, and Bacteroides distasonis were investigated. The minimal concentration of normal human serum that facilitated maximal uptake by human peripheral leukocytes of 14C-labeled strains ranged from 5% to 80%. Heated (56 C at 30 min) serum produced less uptake than untreated serum, suggesting that complement was required for opsonization. Using human serum depleted of C4 by immunoadsorption as the source of opsonins, predominant utilization of the alternative complement pathway during opsonization was established. Acidic polyanionic exopolymer on the strains was also assessed by ruthenium red staining, electron microscopy, and India ink wet mounts. Strains of the species B thetaiotaomicron, B vulgatus, and B fragilis possessed exopolymer, and a unique configuration of this material was observed on two strains that resisted opsonization. Utilization of classical and/or alternative complement pathway activity during opsonization was not related to the presence of exopolymer.

Bacteroides fragilis↗

The role of complement and antibody in opsonophagocytosis of type II group B streptococci.

The role of complement and antibody in the opsonophagocytosis of type II group B streptococci (type II GBS) was defined with sera from healthy adults and two populations with theoretical susceptibility to type II GBS infection--neonates and insulin-dependent diabetics. Significant opsonophagocytosis (bactericidal index, greater than or equal to 90%) of five clinical isolates of type II GBS lacking components of protein antigen c was demonstrated by each of 12 adult sera, as well as by agammaglobulinemic serum, a result indicating that opsonophagocytosis can proceed by antibody-independent activation of the classic complement pathway. Strains containing components of protein antigen c were somewhat more resistant to opsonin-binding activity. Four of 10 neonatal sera and nine of 15 diabetic sera exhibited inefficient opsonophagocytosis. Some of the adult sera with either high or low concentrations of specific antibody to type II GBS promoted opsonophagocytosis via the alternative complement pathway, but this response was not observed with neonatal sera. The addition of sufficient amounts of specific antibody to type II GBS to neonatal and adult diabetic sera in vitro, however, promoted efficient opsonophagocytosis via the alternative pathway.

Adult↗

Type III group B Streptococcus: functional interaction with IgG subclass antibodies.

The functionally active IgG subclasses and the portion of the antibody molecule mediating opsonophagocytosis of type III, group B Streptococcus (III-GBS) have not previously been investigated. Serum from a healthy adult immunized with III-GBS vaccine was subjected to ion exchange chromatography, yielding 2,950 mg of IgG/dl and 1,440 micrograms of III-GBS-specific antibody/ml. Papain digestion for selective cleavage of IgG1 and IgG3 yielded an IgG2-rich pool that contained 31% of the initial IgG but 69% of the antibody to III-GBS. This pool retained opsonophagocytic activity of antibody to III-GBS at levels similar to that for the initial IgG pool. Analysis of F(ab')2 fragments of pepsin-digested IgG revealed that the level of alternative pathway-mediated opsonophagocytosis of III-GBS was similar to that mediating functional activity in the initial IgG preparation. Therefore, IgG2 is an active opsonin for III-GBS, but divalency appears to be required for alternative pathway-mediated opsonophagocytosis.

Antibodies, Bacterial↗

Unopsonized Candida albicans hyphae stimulate a neutrophil respiratory burst and a cytosolic calcium flux without membrane depolarization.

We have defined major differences between events elicited during activation of human neutrophils by opsonized Candida hyphae (noningestible) or zymosan (ingestible) and by unopsonized Candida hyphae (also noningestible). In response to opsonized particles, a transient increase in cytosolic free calcium was initially observed, followed by depolarization in the transmembrane potential and a respiratory burst. Without opsonins, zymosan did not elicit a response from neutrophils. In contrast, unopsonized Candida hyphae elicited a transient increase in cytosolic calcium, as well as triggered a respiratory burst; they did not, however, elicit a depolarization of the neutrophil membrane. Furthermore, the rate of superoxide generation decreased after a prolonged lag period. The calcium flux was also delayed and coincided with the onset of superoxide generation. Thus, our data demonstrate that Candida albicans hyphae activate neutrophils in the absence of added serum components and initiate a neutrophil respiratory burst without an antecedent membrane depolarization.

Calcium↗

IgG subclass response and opsonization of Streptococcus pneumoniae after vaccination of healthy adults.

Studies relating opsonization and IgG antibodies to Streptococcus pneumoniae have yielded contradictory results. This study compared changes in opsonization with IgG subclass response after vaccinating healthy subjects with a 23-valent pneumococcal vaccine. Total IgG and IgG subclass antibodies to pneumococcal polysaccharide types 8, 9, and 19 were measured by ELISA. Opsonic activity was assayed using 3H-labeled bacteria and polymorphonuclear leukocytes in different serum concentrations (5%-40%). A substantial postvaccination increase in total and subclass IgG antibody was observed in most subjects, although variations were seen. Postvaccination sera generally gave rise to enhanced opsonization, and a correlation was found between increases in antibody levels and opsonization. This correlation was closest for IgG1 and IgG4 and generally strongest at the lowest serum concentration, but weak or absent at the highest concentration. Thus, vaccination against S. pneumoniae stimulates a variable increase in specific opsonic activity in health persons that is best demonstrated when serum is a limiting factor in the opsonin assay.

Adult↗

Pathogenesis of intraabdominal abscess formation: abscess-potentiating agents and inhibition of complement-dependent opsonization of abscess-inducing bacteria.

Bacteroides fragilis and Escherichia coli are synergistic in the production of intraabdominal abscesses. However, these bacteria initiate abscess formation only when inoculated with an agent such as autoclaved colonic contents (ACC) or bran (a fiber analogue). The mechanism of action of the abscess-potentiating agent was studied. Opsonins in normal mouse serum were determined for phagocytic killing by murine neutrophils of B. fragilis and E. coli. Opsonization required fixation of complement by the alternative pathway. ACC (0.2 mg/ml) and bran (1.0 mg/ml) inhibited phagocytic killing of Proteus mirabilis in the presence of normal but not immune serum. Assay of the alternative pathway of complement activation indicated that both bacterial components and abscess-potentiating agents in an abscess-inducing mixture activated complement. These findings suggest that abscess-potentiating agents inhibit opsonization and therefore the subsequent phagocytic killing of bacteria in the nonimmune host.

Abscess↗

The role of complement in the host's defense against Haemophilus influenzae.

In vitro studies have shown that Haemophilus influenzae type b (Hib) can activate both the classical and alternative pathways of complement and generate complement-dependent opsonic and bactericidal activities. In vivo studies and observations in complement-deficient patients have established the biologic significance of complement in the host's defense against H. influenzae. The complement system plays a significant role in the host's defense against Hib and against other encapsulated and unencapsulated H. influenzae, mainly by enhancing clearance from the bloodstream through its action as an opsonin in both nonimmune and immune hosts. Patients with genetically determined deficiencies of C3 or of the complement components involved in C3 activation have an increased susceptibility to H. influenzae. More recently, a relatively common deficiency of one isotype of C4 (C4B) has been shown to be associated with invasive Hib disease, suggesting that defects in complement-mediated host defense may be more common in systemic Hib infections than previously appreciated.

Animals↗

Comparison of the opsonic activity of human surfactant protein A for Staphylococcus aureus and Streptococcus pneumoniae with rabbit and human macrophages.

Surfactant protein A (SP-A) is a glycosylated apoprotein that may facilitate bacterial phagocytosis and contribute to early bacterial clearance in the lung. The effect of SP-A on attachment (or ingestion) of Staphylococcus aureus and type 25 pneumococci to rabbit alveolar macrophages and human monocyte-derived macrophages was studied. SP-A bound to S. aureus and type 25 pneumococci in a calcium-dependent manner. Bacteria-associated SP-A significantly increased attachment of S. aureus, but not pneumococci, to macrophages. Increased association of SP-A-coated S. aureus with macrophages appeared to consist mainly of attachment without ingestion, as determined by bactericidal tests and release of tritiated bacterial digestion products from macrophages. Preincubation of macrophages with SP-A did not increase attachment or ingestion of S. aureus or type 25 pneumococci, with or without the addition of immune opsonins. SP-A acts as a ligand to facilitate attachment of S. aureus to macrophages but has no effect on S. pneumoniae.

Animals↗

Opsonization and antibodies to capsular and cell wall polysaccharides of Streptococcus pneumoniae.

Opsonin-dependent phagocytosis is believed to be the major defense mechanism against Streptococcus pneumoniae. In addition to type-specific antibodies to pneumococcal capsular polysaccharides (PPS), most persons also produce antibodies to cell wall polysaccharide (CWPS), which may be detected in antibody assays due to contamination of PPS antigens. Antibodies to CWPS and PPS from common serotypes were analyzed in relation to opsonic activity before and after vaccination of healthy adults. A highly significant relationship was generally found between opsonization and pneumococcal antibody levels, and this correlation increased markedly by neutralization of CWPS antibodies. This applied in particular for IgG2, which showed no correlation without neutralization. Adsorption of PPS antibodies almost abolished the opsonic activity, whereas removal of CWPS antibodies had no effect. These findings indicate that opsonization of pneumococci is almost exclusively mediated by PPS antibodies and emphasize the importance of neutralizing CWPS antibodies when immunity to this organism is evaluated.

Adult↗

Opsonic activities of surfactant proteins A and D in phagocytosis of gram-negative bacteria by alveolar macrophages.

Surfactant proteins A and D (SP-A, SP-D) can interact with lipopolysaccharide (LPS) and stimulate alveolar macrophages. The opsonic activities of SP-A and SP-D for bacteria with different types of LPS and alveolar macrophages were investigated. In flow cytometric studies with fluorescein-labeled rough (J5) and smooth (O111) Escherichia coli and rat alveolar macrophages, SP-A enhanced binding of J5 but not O111 bacteria to macrophages. Most importantly, SP-A enhanced ingestion of J5 bacteria by alveolar macrophages and subsequent bacterial killing. Immunoelectron microscopy demonstrated that J5 bacteria, the interface between the bacterium and the outer membrane of the alveolar macrophage, and ingested bacteria were heavily labeled with SP-A. In contrast, SP-D did not mediate phagocytosis. SP-A acted as an opsonin in the phagocytosis of rough LPS-containing bacteria by alveolar macrophages, emphasizing the possible role for SP-A in the alveolar defense system.

Animals↗

Influence of subinhibitory concentrations of ceftriaxone on opsonization and killing of Pseudomonas aeruginosa by human neutrophils.

Ceftriaxone, a 2-aminothiazolyl cephalosporin does not alter human neutrophil (PMN) bactericidal function. However, low concentrations of ceftriaxone induce some bacterial strains to be more sensitive to PMN killing. We have studied the effect of a subinhibitory concentration of ceftriaxone (10 mg/l) on Pseudomonas aeruginosa (MIC greater than 128 mg/l). After an overnight exposure to this concentration of ceftriaxone, P. aeruginosa elongated into filaments. PMN killing of ceftriaxone-treated bacteria was better than killing of control bacteria. This enhanced killing was correlated with an increased sensitivity to oxygen-dependent bacterial killing. Furthermore, the altered bacteria induced a greater oxidative response of PMN which was independent of their chemiluminescence response after stimulation by control P. aeruginosa. This increased oxidative burst was attributable to both non-opsonodependent stimulation and to increased deposit of opsonins.

Ceftriaxone↗

Prevention of liver reticuloendothelial systemic host defense failure after surgery by intravenous opsonic glycoprotein therapy.

Depression of the reticuloendothelial system (RES) was observed in rats following operative trauma consisting of laparotomy and jejunal enterotomy. This RES depression was manifested as a significant impairment in the phagocytic clearance of intravenously injected blood-borne test colloid mediated by a decline in hepatic Kupffer cell phagocytosis. Reticuloendothelial systemic host defense depression was correlated with a significant decline in bioassayable and immunoreactive opsonic alpha(2)SB glycoprotein concentration over a 1-3 hr postoperative period with rebound elevation in opsonic activity by 24 hr postsurgery. Intravenous administration of purified opsonic alpha(2)SB glycoprotein at the end of the operation prevented postoperative opsonic deficiency and restored normal hepatic RES phagocytic function. These studies coupled with previous observations in patients following surgery or after severe multiple trauma suggest that reticuloendothelial depression during and after operation mediated by opsonic deficiency may lead to a precarious imbalance between systemic host defense and the dissemination of blood-borne foreign and effete particulate matter such as injured platelets, fibrin microaggregates and immune complexes. Immunoreactive serum opsonic alpha(2)SB glycoprotein determinations may serve as a valuable index of hepatic RE clearance capacity and opsonin therapy may potentially be a selective means to augment systemic host defense.

Animals↗

Deficits in reticuloendothelial humoral control mechanisms in patients after trauma.

Plasma opsonic activity as expressed by an alpha-2-globulin which stimulates hepatic Kupffer cell phagocytosis, and thus modulates RES clearance, was determined in patients at varying intervals following whole-body trauma. Plasma opsonic activity decreased markedly following trauma in both nonsurviving (NS) and surviving (S) trauma patients as compared to an age- and sex-matched group of healthy volunteers. The initial post-traumatic hypoopsonemia (0-72 hr) was more severe (p less than 0.01) in nonsurviving patients than surviving patients. Survivors following trauma manifested restoration of opsonin levels with a definite transient rebound hyperopsonemia during the recovery phase (11-30 days); nonsurviving patients exhibited persistent systemic alpha-2-globulin opsonic deficiency. On the basis of previous animal and human studies, the presently observed humoral deficits following trauma in patients could contribute to impairment of reticuloendothelial Kupffer cell clearance of blood-borne particulate matter such as fibrin, damaged platelets, and other altered autologous tissue. The importance of post-trauma RES dysfunction to survival following severe injury warrants further investigation and clinical consideration.

Adolescent↗

Opsonic fibronectin after operation or trauma: effect of hemodilution and duration of operation.

Fibronectin is an essential opsonin for phagocytosis of nonbacterial particulate matter by the reticuloendothelial system (RES). Fibronectin deficiency causes RES phagocytic depression and repletion of fibronectin reverses RES depression. Fibronectin depletion has been linked with multiple organ failure after surgery and trauma in patients with intra-abdominal infection. The mechanisms for fibronectin depletion have not been established. Plasma fibronectin levels were measured in 29 patients requiring operation after trauma and 15 patients undergoing elective abdominal operations. Opsonic fibronectin fell in the immediate postoperative period and on the first postoperative day in both groups. Total fluid replacement, blood loss, and operative time did not correlate with depression in fibronectin levels after trauma, although a weak correlation may exist between total fluid administration and fibronectin concentration in postoperative patients. Total fluid replacement, blood loss, and operative time may be contributing factors, but are not the major determinants of fibronectin deficiency after trauma or operation.

Abdomen↗

Opsonic requirements of Staphylococcus epidermidis.

The opsonic requirements of 18 strains of Staphylococcus epidermidis were compared in pooled normal human serum and in peritoneal dialysate from patients undergoing continuous ambulatory peritoneal dialysis (CAPD). A serum concentration of 2.5% gave optimal opsonisation. The opsonisation of all strains was antibody- and complement-dependent, and there were no significant differences in the pattern of their opsonic requirements. Peritoneal-dialysis effluent from uninfected patients was a poor source of opsonins because of the low levels of immunoglobulin G and of the C3 component of complement it contained. Growth of S. epidermidis in peritoneal-dialysis effluent rather than in broth did not alter its opsonic requirements. Strains from patients undergoing CAPD and suffering from peritonitis were not more resistant to opsonisation and phagocytic killing than those from other sources.

Complement System Proteins↗

Opsonic requirements of Helicobacter pylori.

The opsonic requirements of Helicobacter pylori were investigated in a series of experiments with human polymorphonuclear leucocytes (PMNL). Pre-incubation of H. pylori with pooled normal human serum (NHS) in concentrations of 5-20% significantly increased the uptake of radiolabelled bacteria by PMNL. Treatment of the bacteria with NHS 30% caused the release of radiolabel and this effect was abolished by heating serum to 56 degrees C, suggesting that H. pylori is serum-sensitive and that complement is involved. Opsonisation of H. pylori with NHS concentrations of 10-30% significantly increased PMNL chemiluminescence. Removal of specific antibody had no effect. Removal of either the classical or alternative complement pathways produced no significant change in PMNL chemiluminescence, indicating that either pathway is sufficient for opsonisation on its own. The results confirm that complement is the most efficient opsonin for H. pylori.

Analysis of Variance↗