Assessment of serum factor B, serum opsonins, granulocyte chemotaxis, and infection in nephrotic syndrome of children.
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The opsonization of type 1 and type 25 pneumococci in the rabbit lung early in the course of experimental pneumonia was studied. Bacteria washed from the lungs of animals with type 1 pneumonia were coated with IgG, IgA, and complement within 24 hr of infection; these bacteria were not phagocytized. Both type 1 and type 25 pneumococci, when incubatd in concentrates of nonimmune bronchopulmonary washings (BPC), adsorbed IgG, IgA, and C3, but neither type of pneumococci was opsonized. Killing of pneumococci by surface phagocytosis, with or without the use of nonimmune BPC, was not demonstrated. When nonimmune serum was used, opsonization of type 25 pneumococci depended on the presence of heat-labile adsorbable factors, and when immune serum and immune BPC were used, opsonization was dependent on adsorbable factors. Depletion of serum complement with cobra venom factor before infection with type 25 pneumococci resulted in severe bacteremic pneumonia in the rabbit, but immunization before complement depletion allowed animals to clear type 25 pneumococci from the blood and lungs within 24 hr. Thus, opsonization is required for efficient clearance of pneumococci from the lung, and in the nonimmune animal, complement is also required for clearance.
The interaction of Cryptococcus neoformans with the murine macrophage-like cell line J774.16 was studied in the presence and absence of monoclonal antibodies (MAbs) to the capsular polysaccharide glucuronoxylomannan (GXM). In the absence of MAb 2H1 to GXM, coincubation of J774.16 cells with C. neoformans reduced fungal colony-forming units in only 26.6% of 21 independent experiments. In the presence of MAb 2H1, coincubation of J774.16 cells with C. neoformans reduced fungal colony-forming units in > 95% of experiments. Comparison of the relative efficacy of two IgG1 MAbs revealed that the higher affinity MAb was more effective at low concentrations. Antibody-mediated reductions of C. neoformans colony-forming units by J774.16 cells occurred despite inhibition of nitric oxide synthase, addition of reactive oxygen intermediate scavengers, or the use of a superoxide-deficient J774 mutant line.
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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.
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antisera were prepared in rabbits against whole organisms of colony type 1 Neisseria gonorrhoeae strains F62 and B (fron gonococcal urethritis) and 7122 (a strain typical of those associated with disseminated gonococcal infection), and against purified outer membrane components from the same strains including pili and principal outer membrane protein. Antibody levels to pili, principal outer membrane protein and lipopolysaccharide were determined using a quantitative enzyme-linked immunosorbent assay. Each antiserum was heat-inactivated and tested for opsonic for its homologous strain, and this immune-enhanced phagocytosis was decreased by adsorption with homologous purified outer membrane components: pili greater than lipopolysaccharide greater than principal outer membrane protein. Opsonic activity was approximately equal for antiserum to purified pili and antiserum to the whole organisms for each of the three strains, and purified antibody to pili was highly opsonic. The F(ab')2 fragments of antibody to pili were not opsonic, indicating a role for the Fc receptor on the phagocyte membrane in immune-enhanced phagocytosis of gonococci.
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When sera diluted to 5% in a buffer containing calcium and magnesium were incubated with mannan-coated ELISA plates, C4 fragments, properdin and factor B were bound to the plates as well as the expected opsonic C3 fragments, C3b and C3bi. The calcium-dependent lectin mannan-binding protein, which is structurally similar to C1q, was also shown to bind in this assay and analysis of sera from 179 healthy blood donors revealed that the binding levels of all these proteins were highly significantly correlated. Results obtained with a previously described C3b opsonic assay using zymosan also correlated with the mannan-binding levels. When the sera were diluted to 5% in the presence of Mg-EGTA there was no detectable binding of complement proteins to the mannan surface, confirming that no alternative pathway activation occurred at this serum concentration. When sera were diluted to 5% in a buffer containing EDTA in order to study immunoglobulin binding in the absence of complement activation, the levels of bound IgG1, IgG2, IgG3, IgA and IgM antibodies were found to be completely unrelated to the C3bi binding levels previously observed. The results suggest that in this experimental system using low concentrations of serum, mannan-binding protein initiates an antibody-independent mechanism of cleavage of the classical pathway component C4, which subsequently regulates the degree of cleavage of C3 and recruitment of alternative pathway proteins.
The concentrations of mannan-binding protein (MBP) in consecutive samples from 10 HIV+ persons were estimated using an ELISA based on polyclonal rabbit anti-MBP. The changes in MBP with time were similar in HIV+ and HIV- persons, and did not appear to be of clinical significance. MBP was determined in a further 70 persons found HIV-1+ during a period of 2.5 years (1984-1986). Out of the total of 80 patients, 32 have by now died from AIDS. According to the serum level of MBP the HIV-infected persons were grouped into high (> 650 ng MBP/ml), intermediate (101-650 ng/ml), and low MBP (< 101 ng/ml). At the termination of the study the frequency of deaths/total in each of the groups were: high MBP, 14/39 (36%); intermediate MBP, 12/26 (46%); and low MBP, 6/14 (43%). There was no association between the MBP level of the individual and the progressive loss of CD4+ T cells, and the level of MBP was not predictive for the length of time between the detection of HIV antibodies and development of AIDS, nor for the duration of AIDS before death occurred. The number of HIV+ persons without detectable MBP (10%) was significantly higher than previously reported for healthy persons (2.4%, P = 0.027). The course of HIV infection does not seem to be influenced by the level of MBP, nor does the antimicrobial activity of MBP appear to affect the progression of AIDS. Further studies are required to substantiate the significance of absence of MBP in the susceptibility to HIV.
Sera from a group of randomly selected control children between the ages of one and fourteen years were assessed for opsonic activity utilising the Neutrophil Iodination Micro-Method (NIMM). The opsonic capacity of fresh and heat inactivated serum from these children was compared with that of adult controls utilising the following micro-organisms: C. albicans, S. cerevisiae, zymosan, S. aureus (Oxford and Cowan I strains), S. epidermidis, E. coli meningitidis, and a nonpathogenic strain of E coli. The overall opsonic capacity of children under the age of four was similar to that of the adults and the older children (four to fourteen years) for all the micro-organisms tested except C. albicans. With five of the eight micro-organisms tested the heat stable opsonic activity of children in the younger age group was significantly lower than the other two groups tested. It is suggested that opsonic function in children approaches that of adults around the fourth year of life.
Serum opsonic activity against a strain of group B meningococci was measured by the initial increment in polymorphonuclear leukocyte chemiluminescence. Chemiluminescence was markedly enhanced by serum and was dependent upon both thermostable and thermolabile factors. It was higher in convalescent sera from patients with meningococcal disease than from other patients and normal controls. Absorption of sera with the homologous strain substantially decreased the opsonic activity. Convalescent serum from a patient with group C meningococcal pericarditis showed high opsonic activity towards a group B meningococcal strain. A convalescent serum showed significantly higher activity towards its homologous group B strain than towards another group B meningococcal strain. As measured by chemiluminescence, properties besides group specificity are of importance in opsonization of meningococci. The simplicity and precision of the chemiluminescence measurements seem to offer an improved tool for the study of meningococcal disease immunology.
In an earlier study, with the use of chemiluminescence (CL) and phagocytic killing, we could show that in the presence of serum from healthy adults polymorphonuclear leukocytes (PMNL) efficiently handle nonpathogenic Neisseria meningitidis strains, in sharp contrast to those associated with clinical disease. The major part of this difference was dependent on serum factors. In the present study 84 serum samples from children 1-3, 4-6, 7-9, and 10-14 years old were studied by the CL technique according to their ability to opsonize meningococci. There was a highly significant difference (p less than 0.001) in all four age groups when the CL indexes obtained with the pathogenic meningococci of the serogroups A, B and C were compared with those of the nonpathogenic menigococci: serogroup 29E and nongroupable meningococci. These findings imply that the ability to opsonize so-called nonpathogenic meningococci is developed early in life and may explain why they are only occasionally able to cause disease.
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Uterine washings were found to promote neutrophil mediated killing of Streptococcus zooepidemicus. Depletion of complement and/or specific antibody from the washings significantly reduced bactericidal activity. Phagocytosis of yeast by uterine washings was complement dependent. Inhibition of the classical pathway significantly reduced opsonic activity indicating that, in addition to direct activation via the alternate pathway, antibody may also be involved in yeast phagocytosis.
We suggest a novel approach to enhancing antimicrobial drug action by utilizing engineered peptide conjugates. Our most potent conjugates, [fMLF]PMBN and [fMLF]PMEN, are nonapeptides derived from polymyxin B's (PMB's) cyclic moiety (Thr-Dab-cyclo[Dab-Dab-d-Phe-Leu-Dab-Dab-Thr], where Dab is 2,4-diaminobutyric acid) and polymyxin E's (PME's) cyclic moiety (Thr-Dab-cyclo[Dab-Dab-d-Leu-Leu-Dab-Dab-Thr]), respectively, attached to a linear tail comprised of formyl-Met-Leu-Phe (fMLF). The cyclic part binds to gram-negative lipopolysaccharides, rendering the bacterial outer membrane permeable to hydrophobic antibiotics. The tail confers chemotactic and opsonic activities upon the conjugates. These two activities appear to be the basis for the conjugates' antibacterial activities. The conjugates are 8 to 10 times less toxic than the parent PMB or PME antibiotics. Fourteen of 18 mice lethally challenged with erythromycin-resistant Klebsiella pneumoniae survived following intraperitoneal administration of erythromycin and [fMLF]PMBN, whereas erythromycin or the peptide conjugate alone had no effect. Moreover, the clearance of Klebsiella from blood was markedly enhanced by intravenous injection of the [fMLF]PMEN peptide conjugate compared to the clearance of the organism from the mice treated with buffer alone as a control and was similar to that achieved by the PME antibiotic. Blood clearance was also significantly enhanced by administration of PMEN either alone or in a mixture with fMLF, although the effect was less than that produced by the peptide conjugate. Since resistance to polymyxins, the parent molecules of the synthetic cyclic peptides, is rare, the emergence of bacteria resistant to the antimicrobial properties of the peptide conjugates may be precluded as well.
Several species of mycoplasmas are responsible for respiratory disease in animals and man. As yet, little is known about the interaction of these pathogens with alveolar macrophages, one of the primary components of pulmonary resistance to infections. The present study was undertaken to develop an in vitro model to examine this organism-cell interaction, using a human pathogen, mycoplasma pneumoniae, and normal guinea pig alveolar macrophages. During a 24-h incubation of M. pneumoniae with a monolayer of macrophages, mycoplasmas were found to attach directly to the surface of the cells without inducing significant phagocytosis. Ultrastructurally, the organisms appeared bound to the cell membrane by their characteristic attachment organelles. Only after the addition of specific anti-mycoplasma serum were cells able to engulf attached and surrounding organisms. These data suggest that the interaction of M. pneumoniae and alveolar macrophages is a potentially important aspect of disease pathogenesis, and immune factors which might alter this interaction merit further examination.