Phagocytosis in subacute bacterial endocarditis. Localization of the primary opsonic site to Fc fragment.

The opsonic properties of immune gammaG-globulins isolated from patients with chronic septicemic conditions, principally subacute bacterial endocarditis were studied. Opsonic capacity as well as complement-fixing properties of gamma-globulins appeared to be closely associated with integrity of Fc structures. Progressive pepsin digestion of immune gammaG-globulins, as monitored by successive loss of Gm(a) and Gm(b) antigens, abolished opsonic activity. Colostral gammaA, containing agglutinating antibacterial antibodies but no demonstrable complement-fixing activity, was devoid of opsonic capacity. Reduction of gamma-globulin opsonins with 0.01 or 0.1 M mercaptoethanol progressively abolished opsonic activity in parallel with loss of ability of treated gamma-globulins to fix complement with bacteria. Treatment of gamma-globulin opsonins with 0.01 M sodium metaperiodate also produced complete loss of opsonic capacity in parallel with loss of Gm(b) Fc antigens. These findings, together with antiopsonic effects demonstrable with anti-gamma-globulin factors showing primary reactivity with Fc structures, indicate that the opsonic property of immune gamma-globulins requires the participation of structures integral to the Fc region of gamma-globulin.

The relationship between group A and group C meningococcal polysaccharides and serum opsonins in man.

The interaction in vitro between human granulocytes and meningococci in the presence of sera from volunteers immunized by Gotschlich et al. with purified group A and group C meningococcal polysaccharides was studied. Phagocytosis of meningococci did not occur in the presence of preimmunization sera. In all volunteers tested, group-specific opsonins were detected in groups A and C polysaccharide antisera. Opsonic activity appeared within 1 wk after immunization and persisted for at least 14 months. Titers of opsonic activity ranged from 1:20 to 1:320; highest titers were noted in 2-4 wk antisera. Meningococcal opsonins were detected in both 19S and 7S immunoglobulins. Opsonic activity in low-titer antisera depended on heat-labile factors present in both normal and immune sera, whereas phagocytosis was observed in the presence of heat-inactivated high-titer antisera. Phagocytosis of group A meningococci in the presence of certain group A polysaccharide antisera was inhibited by N-acetyl mannosamine, but not by mannose, mannosamine, N-acetyl glucosamine, N-acetyl galactosamine, or N-acetyl neuraminic acid. Absorption studies with sera from patients with natural meningococcal infections revealed that these polysaccharides are the major antiphagocytic determinants for group A and group C meningococci. These studies are consistent with previous reports suggesting that immunization with group A and group C polysaccharides may well provide group-specific protection against meningococcal infections.

Streptococcal M protein extracted by nonionic detergent. II. Analysis of the antibody response to the multiple antigenic determinants of the M-protein molecule.

Purified streptococcal M protein extracted by nonionic detergent was used in an RIA and a solid-phase radiocompetitive inhibition assay to determine the nature of the immune response in both human beings and hyperimmunized rabbits to this complex antiphagocytic antigen. Results indicate that a type-specific response to an M antigen with the development of opsonic antibodies is the result of antibodies directed against the majority of the antigenic determinants of the molecule. Cross-reactions between certain M types on the other hand, are represented by antibodies directed against only a small percentage of these antigenic determinants. Results also suggest that avidity may play a role in the action of opsonic antibodies. However, the data indicate that factors besides avidity (i.e. sites bound by the antibodies) also seem essential for opsonization.

Serum complement-like opsonic activities in human, animal, vegetable, and proprietary milks.

Human, animal, proprietary, and soy milks are comparable to human serum C5 in opsonization of baker's yeast. Bovine milk and human serum opsonically reconstitute C5-deficient mouse serum. Such reconstitution is selectively inhibited by antiserum to human C5. Further characterization suggests that bovine milk contains material structurally and functionally similar, but not identical, to human C5.

Lung fluid immunoglobulin from HIV-infected subjects has impaired opsonic function against pneumococci.

BACKGROUND: The incidence of pneumococcal pneumonia is greatly increased among human immunodeficiency virus (HIV)-infected subjects, compared with among non-HIV-infected subjects. Lung fluid levels of immunoglobulin G (IgG) specific for pneumococcal capsular polysaccharide are not reduced in HIV-infected subjects; therefore, we examined immunoglobulin subtypes and compared lung fluid IgG opsonic function in HIV-infected subjects with that in healthy subjects. METHODS: Bronchoalveolar lavage (BAL) fluid and serum samples were collected from 23 HIV-infected and 26 uninfected subjects. None of the subjects were receiving highly active antiretroviral therapy, and none had received pneumococcal vaccination. Pneumococcal capsule-specific IgG levels in serum and BAL fluid were measured by enzyme-linked immunosorbent assay, and IgG was concentrated from 40 mL of BAL fluid. Opsonization and opsonophagocytosis of pneumococci with serum, BAL fluid, and BAL IgG were compared between HIV-infected subjects and healthy subjects. RESULTS: The effect of type 1 pneumococcal capsular polysaccharide-specific IgG in opsonizing of pneumococci was significantly less using both serum and BAL IgG from HIV-infected subjects, compared with serum and BAL IgG from healthy subjects (mean level, 8.9 fluorescence units [95% confidence interval, 8.1-9.7 fluorescence units] vs. 12.1 fluorescence units [95% confidence interval, 9.7-15.2 fluorescence units]; P=.002 for lung BAL IgG). The opsonophagocytosis of pneumococci observed using BAL IgG from HIV-infected subjects was significantly less than that observed using BAL IgG from healthy subjects (37 fluorescence units per ng of IgG [95% confidence interval, 25-53 fluorescence units per ng of IgG] vs. 127 fluorescence units per ng of IgG [95% confidence interval, 109-145 fluorescence units per ng of IgG]; P<.001). CONCLUSION: HIV infection is associated with decreased antipneumococcal opsonic function in BAL fluid and serum.

Influence of a deficiency of the second component of complement on the bactericidal activity of neutrophils in vitro.

Serum from three patients with a complete, selective deficiency of the second component of complement (C2) did not promote optimal killing of Staphylococcus aureus, 502A by neutrophilic polymorphonuclear leukocytes (PMN) in vitro. The addition of C2 reagent or the presence of heat-stable opsonin in the C2-deficient serum corrected the defective killing of S. aureus that was observed with patient or control PMN. PMN from the patients or control subjects killed bacteria with equal efficiency under conditions of optimal opsonization (normal pooled serum). However, twice-washed control PMN were better than patient PMN in killing S. aureus under circumstances of suboptimal opsonization (C2-deficient serum, heated C2-deficient serum, heated normal pooled serum, or no replacement of serum). The latter finding was due to residual C2 on the surface of twice-washed control cells. As repeated washing of control PMN progressively removed cell-associated C2, the staphylocidal effectiveness of the control RMN decreased to the level of patient PMN. In contrast to the findings with S. aureus, triply-washed PMN from patients or controls killed normal numbers of Escherichia coli, ON2, in C2-deficient serum.

Proteins of the cystic fibrosis respiratory tract. Fragmented immunoglobulin G opsonic antibody causing defective opsonophagocytosis.

In the disease cystic fibrosis (CF), pulmonary infection with Pseudomonas aeruginosa is a common clinical complication that determines most morbidity and almost all excess mortality. We postulated that in this disease a defect in Pseudomonas-reactive IgG antibodies may contribute to chronic Pseudomonas infections. Bronchoalveolar lavages were performed upon 13 patients with CF, 7 patients with chronic bronchitis characterized by recurrent Pseudomonas infections, and 4 normal volunteers. The levels of various proteins important to host defenses and proteases were determined; enzyme inhibition studies were performed. CF respiratory immunoglobulin levels were significantly elevated when compared with both normals and patients with chronic bronchitis (P less than 0.05). Albumin and transferrin levels were decreased in the CF lung fluids. CF elastolytic activity was strikingly elevated (means = 6.02 micrograms/mg total protein) and the inhibitory profile suggested such activity resembled a serine-proteinase. Alpha-1-antitrypsin antigenic levels were not altered in CF respiratory fluids. There was a tendency for the lavage IgG to fall as elastase levels rose (r = -0.29). IgG opsonins for two Pseudomonas immunotypes were isolated with affinity chromatography for functional and immunochemical studies. Bacterial phagocytic rates in the presence of these Pseudomonas-reactive IgG opsonins derived from CF lavage fluid were depressed (0.3% uptake/unit time) when compared with similarly titered positive controls (uptake = 1.3%/unit time, P less than 0.001). Additionally, normal pulmonary macrophage intracellular killing of Pseudomonas was severely altered in the presence of opsonins derived from CF respiratory fluids. At some time points, less than 30% of the bacteria were killed. CF IgG opsonins contain a cleavage fragment (100,000 D, 5S sedimentation coefficient) with antigenic determinants similar to the Fab portion of IgG. The presence of such a fragment was inversely correlated with phagocytic functional activity. Intact IgG comprised as little as 18% of the CF lavage fluid specimens. Aliquots of intact human IgG, when mixed with the CF opsonins, augmented Pseudomonas uptake and improved intracellular killing. Conversely, peptide fragments of IgG opsonins, which are proteolytically derived in vitro, duplicated in our system the defect observed with opsonins derived from CF lung fluids; bacterial uptake was inversely related to the concentration of F(ab')2 and to a greater degree, to Fc present in the opsonic mixture. We concluded that IgG respiratory opsonins are fragmented, inhibiting phagocytosis and serving a permissive role in the chronic Pseudomonas pulmonary infection in the disease CF.

Time-dependent changes in opsonin amount associated on nanoparticles alter their hepatic uptake characteristics.

The relationship between the time-dependent change in serum proteins adsorbed on nanoparticles and their disposition to the liver was investigated by employing lecithin-coated polystyrene nanosphere with a size of 50 nm (LNS-50) as a model nanoparticle in rats. The total amount of proteins adsorbed on LNS-50 increased and the qualitative profile of serum proteins adsorbed on LNS-50 changed during the incubation with serum up to 360 min. The liver perfusion study indicated that the hepatic uptake of LNS-50 incubated with serum for 360 min was significantly larger than those of LNS-50 incubated for shorter period. It was suggested that the increase in the hepatic uptake of LNS-50 with the increase in incubation time would be ascribed mainly to the increase in the opsonin-mediated uptake by Kupffer cells. Semi-quantification of major opsonins, complement C3 (C3) and immunoglobulin G (IgG), and in vitro uptake study in primary cultured Kupffer cells demonstrated that the increase in C3 and IgG amounts adsorbed on LNS-50 was directly reflected in the increased disposition of LNS-50 to Kupffer cells. These results indicate that the amounts of opsonins associated on nanoparticles would change over time and this process would be substantially reflected in the alteration of their hepatic disposition characteristics.

Modular virus-like particles for sublingual vaccination against group A streptococcus.

Infection with Group A streptococcus (GAS)-an oropharyngeal pathogen-leads to mortality and morbidity, primarily among developing countries and indigenous populations in developed countries. The development of safe and affordable GAS vaccines is challenging, due to the presence of various unique GAS serotypes, antigenic variation within the same serotype, and potential auto-immune responses. In the present study, we evaluated the use of a sublingual freeze-dried (FD) formulation based on immunogenic modular virus-like particles (VLPs) carrying the J8 peptide (J8-VLPs) as a potential safe and cost-effective GAS vaccine for inducing protective systemic and mucosal immunity. By using in vivo tracing of the sublingual J8-VLPs, we visualized the draining of J8-VLPs into the submandibular lymph nodes, in parallel with its rapid absorption into the systemic circulation, which support the induction of effective immune responses in both systemic and mucosal compartments. The sublingual administration of J8-VLPs resulted in a high serum IgG antibody level, with a good balance of Th1 and Th2 immune responses. Of note, sublingual vaccination with J8-VLPs elicited high levels of IgA antibody in the saliva. The co-administration of mucosal adjuvant cholera toxin (CT) further enhanced the increase in salivary IgA antibody levels induced by the J8-VLPs formulation. Moreover, the levels of salivary IgA and serum IgG observed following the administration of the CT-adjuvanted FD formulation of J8-VLPs (FD-J8-VLPs) and non-FD formulation of J8-VLPs were comparable. In fact, the saliva isolated from mice immunized with J8-VLPs and FD-J8-VLPs with CT demonstrated opsonizing activity against GAS in vitro. Thus, we observed that the sublingually delivered FD formulation of microbially produced modular VLPs could prevent and control GAS diseases in endemic areas in a cost-effective manner.

A colorimetric microassay for opsonins by reduction of NBT in phagocytosing bovine polymorphs.

The adaptation of the reduction of nitro blue tetrazolium (NBT) by phagocytes to the measurement of the opsonising capacity of serum or milk whey is described. A dose-response curve of the absorbance of solubilised reduced NBT to the concentration of opsonins, along with a close correlation (r = 0.94) between the colorimetric NBT assay and ingestion of group B streptococci (as measured by reduction of [3H]thymidine incorporation by extracellular bacteria), was established. This NBT reduction test, employing 96-well flat-bottom microtitre plates, is simple, semiautomated, requires a low number of PMN, and is applicable to a large number of samples, which renders it useful as a screening test for the opsonising capacity of sera or other biologic fluids.

Flow cytometric assay for the measurement of serum opsonins to Neisseria meningitidis serogroup B, serotype 15.

A flow cytometric phagocytosis assay has been developed for the measurement of human serum opsonins to serogroup B meningococci. Live bacteria and bacteria inactivated by heat, formalin or ethanol were labelled with fluorescein-isothiocyanate (FITC). The bacteria were opsonized with sera from patients with group B meningococcal disease and sera from healthy controls, and phagocytosis determined by combined measurements of FITC-fluorescence and forward angle light scatter. Optimal sensitivity was obtained using viable bacteria, 5% serum, 20 bacteria per leukocyte capable of phagocytosis, 7.5 min opsonization time, 5 min phagocytosis time, 37 degrees C, and continuous agitation during opsonization and phagocytosis. The opsonic activity of sera from convalescent patients was markedly higher than that of sera from patients with acute illness. Only minor day-to-day and interindividual variations were observed. The flow cytometric phagocytosis technique is a rapid and reproducible method for the measurement of serum opsonins to meningococci.

The use of 7-amino-actinomycin D in the analysis of Candida albicans phagocytosis and opsonization.

We describe the use of 7-amino-actinomycin D (7AAD) to measure phagocytosis and the opsonizing capacity of serum. Heat-inactivated Candida albicans was previously stained with 7AAD and incubated with resident peritoneal macrophages. The samples were analyzed by flow cytometry and phagocytic cells were identified by their bright red fluorescence. This is a rapid, reproducible and reliable one-step procedure and provides a means of evaluating low levels of phagocytosis.

Kinetics of staphylococcal opsonization, attachment, ingestion and killing by human polymorphonuclear leukocytes: a quantitative assay using [3H]thymidine labeled bacteria.

A method has been developed for studying quantitatively the separate processes of bacterial opsonization, phagocytosis, and killing by human polymorphonuclear leukocytes using [3H]thymidine labeled Staphylococcus aureus. Phagocytosis is determined by assaying for leukocytes-associated radioactivity after differential centrifugation and washing the leukocytes. Opsonization is studied by incubating bacteria with an opsonic source for varying durations and then adding leukocytes. By treatment of samples with the muralytic enzyme, lysostaphin, the attachment and ingestion phases of phagocytosis can be separated. Sampling for colony forming units after disruption of the leukocytes permits the measurement of bacterial killing. Using this method, differences in the kinetics of staphylococcal opsonization by normal and C2 deficient sera were defined, opsonic influences on the attachment and ingestion phases of pH agocytosis were delineated, and the influences of different opsonins and leukocyte populations on killing were determined.

Role of opsonins in clinical response to granulocyte transfusion in granulocytopenic patients.

Fifty febrile severely granulocytopenic patients were given four daily transfusions of 2.2 X 10(10) normal donor granulocytes. Twenty-three (46 percent) responded clinically, although both responders and nonresponders were similar in clinical characteristics at the outset. This study examines the relation between serum opsonic activity before initiation of granulocyte administration and clinical response. Opsonic activity to three test organisms (Escherichia coli 286 and ON 2, and Staphylococcus aureus) and to 15 blood stream isolates from 14 patients was measured as serum-dependent uptake of heat-killed 14C-labeled bacteria by normal donor leukopheresis granulocytes in an in vitro assay and compared with results obtained with a standard normal serum in each assay. At a concentration of 8 percent serum, all patient groups were equivalent to standard (90 to 102 percent) for the three test organisms. When rate-limiting concentrations of serum (1 to 2 percent) were employed, opsonic activity remained similar to standard for S. aureus in all patient groups and for the two E. coli strains in responders (82 to 98 percent). In contrast, opsonins for E. coli decreased to 41 to 50 percent of standard in nonresponders (p less than 0.01). When patients with proved infection were separately analyzed, opsonin activity for E. coli 286 and ON 2 was significantly greater in responders than nonresponders (73.6 versus 34.9 percent and 124.8 versus 58.1 percent, respectively for the two strains) (p less than 0.01). Patients with opsonin activity of 50 percent or greater of standard had a greater response rate (73 versus 19 percent and 45 versus 0 percent for the two E. coli strains) (p less than 0.005 and p = 0.08, respectively). Eight of 10 patients with 75 percent or greater of standard for opsonic activity against their own blood stream isolates also responded, whereas zero of four with opsonins less than 75 percent of standard had a favorable outcome. These results indicate that serum opsonic activity may be a determinant of clinical response to granulocyte transfusion in infected granulocytopenic patients and may be predictive of outcome. We conclude that opsonic activity should be assessed in such patients before granulocyte administration and suggest a trial of plasma infusion in opsonin-deficient patients.

Human immunoglobulins for intravenous use: comparison of available preparations for group B streptococcal antibody levels, opsonic activity, and efficacy in animal models.

Currently available human immunoglobulin preparations for intravenous use (IVIGs) are being used (with antibiotics) by some physicians for therapy of sepsis in newborns. Most neonatal sepsis and/or meningitis in this country is caused by group B Streptococcus (GBS), and most of these cases are due to type III GBS (III-GBS). The killing of III-GBS in vitro is dependent on specific IgG antibody. Adequate serum levels of specific III-GBS antibody protect the exposed newborn from the development of invasive disease. Therefore, III-GBS was used as a model to evaluate the activity of three IVIG preparations available for clinical use. Specific antibody levels, in vitro opsonophagocytic killing, and protective efficacy in animal models revealed differences in activity for III-GBS between the three IVIG preparations as well as between IVIG lots from the same manufacturer. Furthermore, it was found that the effect of IVIG using one of the assay methods may not reliably predict activity obtained using the other assays. These data document the inability to predict functional activity against a specific pathogen such as GBS on the part of a lot of IVIG chosen at random. In view of these findings and of the limited data evaluating clinical efficacy, IVIG cannot be recommended at this time for use in the therapy of infectious diseases such as neonatal sepsis.

Non-respiratory haemolymph proteins in the vineyard snail Helix pomatia. Changes after phagocytosis in vivo.

Haemolymph proteins from the vineyard snail Helix pomatia were studied by chromatographic and immune-electrophoretic techniques with the dual purpose of characterizing the normal composition of haemolymph and to look for possible opsonins. The oxygen-carrying proteins, alpha- and beta-haemocyanin, were by far the most abundant proteins, but at least three non-respiratory proteins could be demonstrated. We found consistent changes in the appearance of the immunoprecipitation pattern of these non-respiratory proteins after the injection of particulate foreign matter into the snail's circulation, and we suggest that they may be opsonins. We also found a haemagglutinin in haemolymph. It was present in very low concentration and was similar to, but not identical with the haemagglutinin which is present in the albumin gland.

Immunization against serogroup B meningococci. Opsonin response in vaccinees as measured by chemiluminescence.

One hundred and thirteen healthy volunteers were immunized twice (six weeks apart) with four different doses (12.5, 25, 50 and 100 micrograms, measured as protein content) of an outer membrane vesicle vaccine from a serogroup B meningococcal strain (44/76, B:15:P1.16) complexed to serogroup C meningococcal polysaccharide and/or Al(OH)3 i.e. 12 different vaccines. Serum opsonic activity against the serogroup B strain was measured using a chemiluminescence method. A significant rise in serum opsonic activity was demonstrated in 84 volunteers (74%) six weeks after the first injection and in 97 (86%) six weeks after the second. All vaccinees with low preimmunization values (less than 25 mVs) experienced a significant increase in opsonic activity. A dose-related response was most evident for the vaccines containing adjuvant, and these vaccines were associated with a maximum response six weeks after the second injection, while the vaccines without Al(OH)3 induced a peak response six weeks after the first injection. The postimmunization opsonic activity was similar to that found in convalescent sera, indicating that the vaccines may protect against serogroup B meningococcal disease.

Opsonic fibronectin deficiency in patients with intra-abdominal infection.

The reticuloendothelial system provides host defense functions by the intravascular phagocytosis of bacteria and nonbacterial particulates. Fibronectin is opsonic for reticuloendothelial phagocytosis. Plasma fibronectin was measured before and after operation in patients with intra-abdominal infection. Preoperatively opsonic fibronectin was reduced by 39% of normal control levels in 16 patients with intra-abdominal infection. There was an even greater reduction of opsonic fibronectin after operation that was first observed in the recovery room. This deficiency persisted for the first 4 days with a tendency toward recovery of normal circulating levels by the fifth postoperative day. In contrast, patients who underwent elective major abdominal operation without infection manifested a transient opsonic fibronectin deficiency with recovery by the second and third postoperative days. Eight of 16 patients with intra-abdominal infection developed multiple organ failure. The opsonic fibronectin levels in those patients were lower than the levels in eight patients who did not develop multiple organ failure. Furthermore, there was no tendency toward recovery of normal circulating opsonic fibronectin in those patients. On all days when multiple organ failure occurred there was a marked deficiency of circulating opsonic fibronectin. We conclude that transient opsonic fibronectin deficiency occurs after major elective abdominal operation. Patients with intra-abdominal infection manifest opsonic fibronectin deficiency before operation, and further depletion of opsonic fibronectin occurs after operation. Postoperative multiple organ failure occurs only in association with severe opsonic fibronectin deficiency.

Cardiovascular hemodynamics after opsonic alpha-2-surface binding glycoprotein therapy in injured patients.

Depression of reticuloendothelial (RE) phagocytic function has been clearly documented following trauma and operation. This phagocytic failure is mediated in part by depletion of an opsonic glycoprotein. Depletion of this opsonic protein may result in prolonged blood retention of potentially harmful particulates that may interfere with the microcirculation and may possibly result in altered organ function. Isolation and identification of this opsonic protein has led to the finding of the identity between opsonic glycoprotein and cold insoluble globulin (CIg) or so-called plasma fibronectin. Since CIg is concentrated in cryoprecipitate, this blood component was used as a readily available source of opsonic protein for replacement studies. Nine patients were studied following a 1-hour infusion of cryoprecipitate obtained from 10 units of plasma and suspended in a volume of 250 ml. Both the pulmonary shunt fraction and the fraction of dead space ventilation decreased significantly (P = 0.02) after cryoprecipitate administration. Limb blood flow (P = 0.001), limb oxygen consumption (P = 0.001), and reactive hyperemia of the limb (P = 0.05) increased significantly following cryoprecipitate infusion. Cardiac output, total oxygen consumption did not change consistently. The data demonstrate that the infusion of cryoprecipitate resulted in improved pulmonary and microcirculatory function--possibly due to opsonic glycoprotein replacement.

Hepatic reticuloendothelial host defense failure following surgical trauma.

We evaluated potential pituitary-adrenal influences on postoperative reticuloendothelial (RE) function. Male dogs, 13 to 18 kg (28.6 to 39.6 lb), were used and the operative procedure was hemicolectomy. The RE function was evaluated by a colloid clearance technique and circulating opsonin levels were quantified by bioassay. Normal animals manifested RE depression three hours following incision (49 percent decline in the global phagocytic index K and an associated 30 percent decline in the circulating opsonic activity). Dexamethasone sodium phosphate pretreatment over a three-day preoperative period prevented the postoperative RE clearance failure (control K = 0.69; postoperative k = 0.79) however, a slight (18 percent) but not significant decrease in opsonic activity occurred. Cortisone acetate or adrenocorticotropic hormone over a wide-dosage range manifested no depressing effect on in vitro phagocytosis. These studies, in conjunction with our previous findings of Kupffer cell activation following adrenalectomy, as well as the demonstration of opsonin depletion and RE phagocytic depression following surgery in the absence of the adrenal glands suggest that the pituitary-adrenal system modulates postoperative RE phagocytosis.