Inhibition of complement activation by a secreted Staphylococcus aureus protein.

Staphylococcus aureus can cause a variety of acute and chronic diseases. The ability of S. aureus to cause persistent infections has been linked to its ability to evade or inactivate host immune responses. We have identified a secreted 19-kDa protein produced by S. aureus that binds to the complement protein C3. N-terminal sequencing of this protein identified it as the extracellular fibrinogen-binding protein (Efb). In this study, we demonstrate that Efb can bind to the alpha -chain of C3 and inhibit both the classical and alternative pathways of complement activation. In addition, we show that Efb can inhibit complement-mediated opsonophagocytosis in a dose-dependent manner and that Efb inhibits complement activity by blocking deposition of C3 or by preventing further complement activation beyond C3b. These data suggest that Efb is a virulence factor involved in facilitating persistent S. aureus infections by interfering with complement activity in vivo.

Interactions of liposomes with cells in vitro and in vivo: opsonins and receptors.

A number of studies have appeared recently on the underlying mechanisms of liposome-cell interactions under in vitro conditions, in which isolated cell populations or cell lines were used. However, our knowledge of how liposomes interact with cells and the parameters that influence this in vivo is limited. We will summarize and discuss the relevant studies on this matter in this article. In addition, researchers in this field have long been aware of the interaction of liposomes with blood (or serum/plasma) proteins in vivo and their potential role in the process of the clearance of liposomes from the circulation. Some of the 'opsonizing' proteins, such as complement components, immunoglobulins, which enhance the interactions of liposomes with 'phagocytic cells' have been identified. However, the issue of which types of opsonins determine the fate of liposomes in vivo and how liposomal physicochemical properties such as size, charge and fluidity play an important role in the process of liposome clearance is not clear. Our own observations of one of opsonins, complement component are reviewed herein. As opposed to the fate of conventional liposomes, we briefly touch on the interaction of surface-modified liposomes, which are designed to avoid interactions with blood proteins and/or cells (sterically stabilized liposomes, long-circulating liposomes) and to actively target specific cells or tissues (targeted liposomes: immunoliposomes). Blood proteins such as opsonins are not usually thought to play an important role in the clearance of such liposomes.

[Several aspects of the treatment of suppurative-septic complications with regard to the state of the immune system]. / Nekotorye aspekty lecheniia gnoino-septicheskikh oslozhnenii s uchetom sostoianiia sistemy immuniteta.

A total of 118 patients with grave combined injuries, massive blood loss, and peritonitis of different origin were examined and treated. Monitoring of clinical and immunological parameters showed their correlation with the severity of the clinical status. Assessment of the effects of antibiotics on the opsonophagocytic system of immunity helped select adequate antibacterial therapy and timely correct the treatment, thus notably improving the results.

Monoclonal IgG antibodies influence the migration patterns of lymphocytes in vivo.

Monoclonal antibodies (MoAb) are useful therapeutic agents for the treatment of a variety of human disorders, although the effector mechanisms responsible for the outcome of an efficient immunotherapy remain unclear. This study was designed to address the early effects of MoAb on the migration patterns of lymphocytes in vivo. The clearance profiles and tissue distribution of 111In-labelled rat lymph node cells were examined in both normal and decomplemented allogeneic and semi-allogeneic recipients pre-injected with IgG2b (R3/13) or IgG2a (R2/15S) MoAb directed against the RT1Aa, the classical class I major histocompatibility complex antigen of the DA rat. Both MoAb were equally effective in not only augmenting the removal of DA and (DA x PVG)F1 cells from the circulation and promoting their subsequent localization within the liver but also causing a significant degree of cell lysis during the early phase of cell clearance, even in decomplemented recipients. Although R3/13 and R2/15S are known to target erythrocytes differently in normal and cobra venom factor (CVF)-treated animals, no differences were observed in the migration behaviour of lymph node cells in allogeneic or semi-allogeneic hosts pre-injected with the same MoAb. Since rat lymphocytes express a much higher level of the RT1Aa antigen as compared with erythrocytes, we could not exclude a possible role of residual complement components in the circulation of CVF-treated rats that may have accounted for the observed antibody-dependent effects on target lymphocytes. On the basis of these findings we believe that the design and methodology employed in our present experimental opsonization system were inadequate to define clearly the mechanisms responsible for antibody-mediated removal and destruction of target lymphocytes in vivo.

Effects of growth hormone and insulin-like growth factor I on opsonin receptor expression on local and systemic phagocytes in a lethal peritonitis model.

OBJECTIVE: To investigate the effects of pretreatment with growth hormone (GH) and insulin-like growth factor I (IGF-I) on phagocyte exudation and bacterial clearance, focusing on CD11b and CD32/CD16 expression on local and systemic phagocytes, in a lethal peritonitis model. DESIGN: Prospective randomized experimental study. SETTING: Research laboratory in a university hospital. SUBJECTS: Balb/c mice (n = 21). INTERVENTIONS: Mice were challenged intraperitoneally with 1 x 10(8) Escherichia coli, after 6 days of pretreatment with saline (control), GH (4.8 mg/kg/day), or IGF-I (24 mg/kg/day). Samples were harvested at 4 hrs after the challenge. MEASUREMENTS AND MAIN RESULTS: Viable bacterial counts in peritoneal lavage fluid (PLF) and blood were determined. Peritoneal exudative cells and peripheral blood leukocytes were counted and analyzed for receptor expressions by flow cytometry. GH reduced viable bacterial counts in PLF, as compared with the saline control. GH (three-fold) and IGF-I (two-fold) increased the number of peritoneal exudative neutrophils (PENs), as compared with the saline control. The number of PENs showed an inverse correlation with PLF viable bacterial counts. By contrast, there were no differences in peripheral blood neutrophil (PN) counts among the three groups, nor was there a correlation between PN and PEN counts. CD11b expression was greater on PENs than on PNs in all three groups. CD11b expression on PNs did not differ among the three groups. However, GH increased CD11b expression on PENs, as compared with saline and IGF-I, and this expression showed a positive correlation with PEN numbers and an inverse correlation with PLF viable bacterial counts. CD11b expression on peritoneal macrophages and peripheral blood monocytes did not differ among the three groups. There were no differences in phagocyte CD32/CD16 expression among the three groups. CONCLUSIONS: GH pretreatment enhanced CD11b expression on PENs, but not PNs, possibly in association with enhanced neutrophil recruitment, phagocytosis, and bacterial elimination by PENs, without activation of PNs. GH prophylaxis may be useful for reducing the frequency rate and severity of septic complications, via modulation of CD11b expression on local and systemic neutrophils.

Does the appearance of drug resistance during therapy alter bacterial susceptibility to opsonophagocytosis?

Coagulase-negative staphylococci (CNS) are common causes of infection in patients undergoing chronic ambulatory peritoneal dialysis (CAPD). Their ability to survive intracellularly within peritoneal macrophages and to persist within the peritoneum during antibiotic therapy has led to the development of drug resistance during treatment. Strains of Staphylococcus epidermidis (SE) and Staphytococcus haemolyticus (SH) have been isolated from patients with CAPD during treatment with ciprofloxacin. The respective MIC values pre-and post-therapy were SE-0.25 and 128 mg/L and SH-0.50 and 64 mg/L. The susceptibility of each isolate to opsonophagocytosis was measured in vitro using isolated polymorphonuclear leucocytes (PMN) derived from fresh human blood donations. The bacteria were radiolabelled during growth, opsonised in either 1 or 10% serum and their uptake measured No differences were seen between the pre- and post therapy isolates when using 10% serum as opsonic source (18 vs. 21%); with 1% serum the corresponding values were lower (5 and 8% respectively). Similarly their ability to generate a respiratory burst as measured by chemiluminescence (CL) in the phagocytic cells was not diminished in the strains which had developed resistance to ciprofloxacin. The mean CL response to the strains isolated at outset of therapy ranged from 0.35-0.45 cpsc, and to the resistant strains following therapy from 0.36-0.50 cpsc. It is clear from the present investigation that although the bacterial strain became at least 10 times more resistant to ciprofloxacin during therapy, no change in their susceptibility to phagocytosis occurred refuting the idea that the emergence of drug resistant strains during therapy results in "super-bugs" of greater virulence.

Biocompatibility of a 1.1% amino acid-containing peritoneal dialysis fluid compared to a 2.27% glucose-based peritoneal dialysis fluid.

The biocompatibility of a 1.1% amino acid-containing peritoneal dialysis fluid (AA-PDF) was compared to that of a 2.27% glucose-based peritoneal dialysis fluid (G-PDF). Peritoneal macrophages (PMO), isolated from the peritoneal dialysis (PD) effluents of 10 chronic ambulatory PD patients, were tested for their phagocytosis capacity and peak chemiluminescence response. A subset of PMO was cultured for 24 h with and without lipopolysaccharide (LPS) to study the release of interleukin-1 beta (IL-1 beta) and 8 (IL-8). As control, the interleukin release by blood monocytes of healthy donors was tested. The opsonic activity of the PD effluent was tested as well. Compared to PMO isolated from G-PDF, PMO from AA-PDF showed a significantly better phagocytosis capacity. There was no difference in the peak chemiluminescence response between PMO from AA-PDF and G-PDF. The release of IL-1 beta by unstimulated PMO isolated from the two fluids did not differ. Compared to control monocytes, however, PMO from both fluids showed a considerable spontaneous release of IL-1 beta. When stimulated with LPS, IL-1 beta production by PMO from G-PDF exceeded that of PMO from AA-PDF (p < 0.002). The release of IL-8 by PMO from G-PDF was significantly higher in comparison with PMO from AA-PDF, both spontaneously and after stimulation with LPS (p < 0.02). The opsonic activity of undiluted and to 75% diluted effluents was significantly higher for G-PDF than for AA-PDF (p < 0.01). Thus, compared to the regularly used G-PDF, the phagocytosis capacity as measure for PMO function seems to be better preserved after in vivo exposure to AA-PDF. In addition, the higher release of IL-1 beta and IL-8 by PMO isolated from G-PDF suggests a stronger intra-abdominal activation of PMO, with G-PDF acting as a chemical inflammatory agent. Whether the lower opsonic activity of the AA-PDF is more important for biocompatibility than the other parameters is not clear. Therefore, it is concluded that, although macrophage function is better preserved, it is not proven that the 1.1% AA-PDF studied has an improved biocompatibility compared to 2.27% G-PDF.

Superoxide anion release by alveolar macrophages exposed to respirable industrial fibres: modifying effect of fibre opsonisation.

Phagocyte-derived free radicals are considered to play a role in fibre-related pathology and the components of the lung lining fluid could modify the surface of fibres. Therefore we examined the ability of long amosite asbestos and a range of man-made fibres to stimulate release of superoxide anion from rat alveolar macrophages when they were in their native form (unopsonised) and opsonised by incubation in rat Immunoglobulin G. We also assessed the specific amount of opsonin adsorbed to each fibre type. In the uncoated form all of the fibres produced modest amounts of superoxide release from macrophages. When they were opsonised however there was an effect on stimulation of release of superoxide that was fibre-specific. Both MMVF21 and RCF 1 were dramatically enhanced in their ability to stimulate release and this was related to a high affinity of their surface for IgG. Code 100/475 and SiC were not substantially affected by opsonisation and this was reflected in their low affinity for IgG. Long amosite had low affinity for IgG but showed dramatic enhancement of capacity to stimulate superoxide release. These fibre-specific differences in the effect of a coating of material that is found in the lung lining points out the problems of interpretation of in vitro data and more work on this important area is warranted.

Protective effects of a novel 32-amino acid C-terminal fragment of CAP18 in endotoxemic pigs.

BACKGROUND: Cationic antimicrobial protein of 18 kd (CAP18) is a neutrophil-derived peptide that binds lipopolysaccharide (LPS) with high affinity. We hypothesized that CAP18(106-137), a novel synthetic 32-amino acid C-terminal fragment of CAP18, would neutralize the physiologic derangements induced by LPS in anesthetized swine. METHODS: Pigs were randomly allocated into three groups. Those in the LPS group (n = 6) were infused with LPS (3 micrograms/kg/hr for 4 hours). Pigs in the LPS/CAP18 group (n = 6) were challenged with LPS (3 micrograms/kg/hr for 4 hours) and also treated with CAP18(106-137) (4 mg/kg/hr for 4 hours). Pigs in the RL group (n = 4) received neither LPS nor CAP18(106-137). RESULTS: Treatment with CAP18(106-137) blocked LPS-induced increases in plasma levels of 6-keto-prostaglandin F1 alpha and tumor necrosis factor-alpha and prevented LPS-induced changes in cardiac output, arterial PO2, phagocyte activation, and peripheral leukocyte count. Changes in circulating concentrations of thromboxane B2, mean pulmonary artery pressure, and dynamic pulmonary compliance were attenuated in the LPS/CAP18 group. CONCLUSIONS: Treatment with CAP18(106-137) neutralizes many of the deleterious effects of LPS in pigs.

Degradation of humoral host defense by Candida albicans proteinase.

The effect of an extracellular proteinase from the pathogenic yeast Candida albicans on the bactericidal and opsonizing activities of human serum was studied. The ability of human polymorphonuclear leukocytes to kill Staphylococcus aureus was greatly reduced when the bacteria were opsonized with human serum treated with the proteinase. The reduction in the opsonizing activity of human serum was attributed to degradation of the Fc portion of immunoglobulin G by the action of C. albicans proteinase as determined by immunoprecipitation reaction. However, the Fab portion of immunoglobulin G was resistant to proteolysis by the proteinase. A clear reduction in the bactericidal activity of human serum against Escherichia coli was observed when the serum was treated with C. albicans proteinase. The reduction of serum bactericidal activity was attributed to the degradation of complement C3 by proteolysis by the proteinase as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, while C5 resisted the action of the proteinase. As determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the proteinase also degrades endogenous proteinase inhibitors, such as alpha 2 macroglobulin and alpha 1 proteinase inhibitor, which are involved in regulating inflammation. These results suggest that destruction of a host's defense-oriented or regulatory proteins facilitates debilitation of the infected host.

Opsonic activity of commercially available standard intravenous immunoglobulin preparations.

Several standard intravenous immunoglobulin G (IVIG) products are available in the United States and have been used with the intent to treat or prevent infections in neonates. We evaluated more than 100 lots of IVIG, from 6 products, to determine the amount of opsonic antibody against neonatal pathogens. Neutrophil-mediated opsonophagocytosis was used to determine opsonic activity in these preparations for Staphylococcus epidermidis; Haemophilus influenzae type b; Streptococcus pneumoniae serotypes 3, 14 and 19; Group B Streptococcus serotypes Ia, Ib, Ia/c, II and III; and Escherichia coli (K1). Pathogen-specific opsonic activity of the lots tested ranged from undetectable to 1:80 and was detectable in < 10% to > 90% of lots tested depending on the organism and manufacturer. Within an IVIG lot there was variable opsonic activity against different strains or serotypes of the same organism. Opsonic activity was significantly (P < or = 0.05) affected by the manufacturer's donor pool and less so by the manufacturing method. We conclude that the pathogen-specific opsonic antibody activity of an IVIG lot is: (1) highly variable for several common neonatal pathogens; (2) predominantly dependent on the donor pool and not the manufacturing method. Clinicians may more appropriately select therapy if the pathogen-specific antibody content of IVIG products by lot are known. In the future neonatal IVIG research should focus on using preparations with known pathogen-specific antibody activity.

Effect of anaerobiosis and sulfide on killing of bacteria by polymorphonuclear leukocytes.

Anaerobic microorganisms in periodontal pockets produce toxic amounts of hydrogen sulfide. The capacity of polymorphonuclear leukocytes to kill a capsulated and a non-capsulated variant of a group B streptococcal strain was studied in presence and absence of sulfide. The killing was equally efficient under aerobic and anaerobic conditions. However, in presence of sulfide the killing of the capsulated variant of the strain was significantly inhibited. Since this strain required higher serum concentrations to be killed by the polymorphonuclear leukocytes, it suggested that sulfide interfered with the opsonization of the bacteria. The capacity of sulfide to split the disulfide bonds of complement factor 3 and immunoglobulin G, deposited on the bacterial surface, was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. There was no detectable effect of 2 mM sulfide on immunoglobulin G. However, sulfide released from opsonized bacteria the beta-chain of C3b C3bi, and the C-terminal part of the alpha-chain of C3bi. This region of the alpha-chain of C3bi has been suggested to bind to the complement receptor 3 of polymorphonuclear leukocytes. The beta-chain of C3b/C3bi may augment the binding of opsonized bacteria to the complement receptors of polymorphonuclear leukocytes. The formation of sulfide by the microflora of the periodontal pockets may provide conditions for the bacteria to escape important parts of the host immune system.

[The response of human lymphocytes to Staphylococcus aureus peptidoglycan in a system of opsonic cooperation]. / Otvet limfotsitov cheloveka na peptidoglikan Staphylococcus aureus v sisteme opsonicheskoi kooperatsii.

Interaction of lymphocytes with S. aureus peptidoglycan treated with normal human serum and its fractions was studied on the basis of luminol-dependent chemiluminescent reaction. Treatment with whole serum led to the considerable increase and acceleration of lymphocytic reactions. The opsonic effect mainly depended on antibodies and complement, the contribution of other opsonins (which could be partially attributed to fibronectin) did not exceed 30%. IgG and fibronectin in concentrations, similar to their concentration in normal human plasma, enhanced the reactions 3.4 +/- 0.3 (p less than 0.05) and 1.5 +/- 0.005 (p greater than 0.05) times, respectively. The problem of the intrapopulation profile of opsonic-lymphocytic reactions and their role in the system of cell-mediated and humoral interactions is discussed.

Stimulation of human neutrophil phagocytosis of Candida albicans by a beta-glucan synthase inhibitor, cilofungin.

The effect of a lipopeptide antifungal agent, cilofungin, on serum opsonization and phagocytosis of Candida albicans yeast phase cells in human neutrophil monolayer assays was investigated. Simultaneous addition of fungicidal concentrations of cilofungin did not enhance or inhibit phagocytosis of C. albicans. Pretreatment of Candida blastospores with cilofungin in the absence of serum complement for 1 h did not affect phagocytosis. However, pretreatment of blastospores with cilofungin and complement promoted a significant increase in ingestion. Pretreatment of neutrophils with cilofungin in serum-free media did not affect neutrophil viability. In contrast, pre-exposure of neutrophils to cilofungin in the presence of complement inhibited ingestion of blastospores.

[The chemiluminescence of the polymorphonuclear leukocytes in food poisoning]. / Khemiliuminestsentsiia polimorfnoiadernykh leikotsitov pri pishchevykh toksikoinfektsiiakh.

Luminol- and lucigenin-dependent chemiluminescence (CL) induced by zymosan, opsonized autoserum was used to study the oxygen-dependent bactericidal system of polymorphonuclear leukocytes (PNL) in patients with food toxico-infections, at different disease periods. Different modifications of CL were established to have analogous dynamics on days 1 and 2 of the disease and differences as to the period of convalescence. A correlation was revealed between the amplitude of CL and the intensity of the intoxication syndrome. The conclusion is made about the role played by granulocytes in the disease pathogenesis. The authors review potential mechanisms of participation of PNL free radicals in the pathophysiological shifts responsible for the development, course and outcome of food toxico-infections.