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PubMedDate not supplied

Flow cytometry for the study of phagocyte functions.

Phagocytes play a key role in host defense, and cell defects are associated with increased susceptibility to infection. Flow cytometry offers rapid and reproducible measurements of single cells in suspension and, following staining with one or more fluorochromes, simultaneous examination of several cell functions. Subpopulations of cells can be identified and sorted for morphologic, biochemical, and functional examination, and specially adapted computer systems allow storage of data for subsequent detailed analysis. Several flow-cytometric assays for the study of phagocytes and their interactions with microorganisms have been developed. These assays facilitate the study of (1) phagocyte surface receptors and regulatory molecules; (2) membrane potential; (3) phagocytosis of microorganisms, including the discrimination between attachment to the phagocyte surface and actual internalization; (4) phagosomal pH; (5) degranulation and enzymatic activity; (6) intracellular calcium; (7) oxidative metabolism; (8) intracellular killing of microorganisms; (9) degradation of microorganisms; and (10) exocytosis. In addition, the influence of serum opsonins on phagocyte-microorganism interactions can be studied. Flow-cytometric techniques are applicable to both experimental and clinical work.

Cell Separation↗
PubMedDate not supplied

Current concepts in the management of peritonitis in patients undergoing continuous ambulatory peritoneal dialysis.

During the past five years, continuous ambulatory peritoneal dialysis (CAPD) has become recognized as a major form of therapy for end-stage renal disease. Despite continued advances, peritonitis remains a major limiting factor in the widespread application of CAPD. The pathogenesis of CAPD peritonitis is strikingly different from that of surgical peritonitis. Not only are the sources of infection and the microbiologic findings dissimilar, but the severity of infection is markedly different. Moreover, in CAPD peritonitis, both biofilms (extracellular slime substances) associated with microbial growth on the implanted silicone catheter and compromised local defenses (depletion of opsonins and intracellular survival of certain microorganisms) are of pathogenic importance. Once the diagnosis of peritonitis is suspected, therapy should be instituted immediately. However, the optimal antibiotic regimen and the best route, dose, and duration of antibiotic administration remain unknown because of a paucity of randomized, controlled studies. New approaches to the prevention and treatment of CAPD peritonitis are being evaluated; if this infection can be successfully managed, CAPD promises to become increasingly popular for the treatment of chronic renal failure.

Anti-Bacterial Agents↗
PubMedDate not supplied

Fibronectin: an enhancer of phagocyte function.

Although fibronectin does not act as a direct opsonin as originally described, it does mediate important interactions with phagocytes throughout the inflammatory process. Fragments of fibronectin released by neutrophil proteases are chemoattractants for monocytes. Moreover, subendothelial matrix fibronectin and "inflammatory" fibronectin released from phagocytes enhance phagocyte adherence to host tissues at an inflammatory site. Fibronectin also increases chemokinesis. Once the phagocyte enters tissues, fibronectin promotes C3- and Fc-receptor activation, allowing complement-coated bacteria to be ingested. Finally, fibronectin enhances the bactericidal activity of monocytes. Thus, fibronectin plays an important, but indirect role, in the activities of phagocytes.

Bacteria↗
PubMed1996

Characterization of two mannose-binding protein cDNAs from rhesus monkey (Macaca mulatta): structure and evolutionary implications.

Mannose-binding proteins (MBPs), members of the collectin family, have been implicated as lectin opsonins for various viruses and bacteria. Two distinct but related MBPs, MBP-A and MBP-C, with approximately 55% identity at the amino acid level, have been previously characterized from rodents. In humans, however, only one form of MBP has been characterized. In this paper we report studies elucidating the evolution of primate MBPs. ELISA and Western blot analyses indicated that rhesus and cynomolgus monkeys have two forms of MBP in their sera, while chimpanzees have only one form, similar to humans. Two distinct MBP cDNA clones were isolated and characterized from a rhesus monkey liver cDNA library. Rhesus MBP-A is closely related to the mouse and rat MBP-A, showing 77% and 75% identity at the amino acid level, respectively. Rhesus MBP-A also has three cysteines at the N-terminus, similar to mouse and rat MBP-A and human MBP. Rhesus MBP-C shares 90% identity with the human MBP at the amino acid level and has three cysteines at the N-terminus, in contrast to two cysteine residues found in rodent MBP-C. A stretch of nine amino acids close to the N-terminus, absent in both mouse and rat MBP-A, but present in rodent MBP-C, chicken and human MBPs, is also found in the rhesus MBP-A. The phylogenetic analysis of rhesus and other mammalian MBPs, coupled with the serological data suggest that at least two distinct MBP genes existed prior to mammalian radiation and the hominoid ancestor apparently lost one of these genes or failed to express it.

Amino Acid Sequence↗
PubMed2005

The 'involution' of mannose-binding lectin.

Mannose-binding lectin (MBL) acts as a serum opsonin in innate immune defense and induces complement activation by the lectin pathway. In humans, low levels of functional serum MBL are caused by the dominant action of three single nucleotide substitutions in exon 1 that disrupt the glycine-rich backbone structure of the protein. The presence of common MBL variant alleles is associated with both infectious and autoimmune diseases. Conversely, it has also been suggested that MBL variants are maintained because of selective advantages for the host. In man, the MBL genetic system comprises one functional gene (MBL2) and one expressed pseudogene (MBL1P1), whereas the lower primate, the rhesus monkey resembles rodents with two functional MBL genes. We have investigated the molecular mechanisms behind the evolutionary loss of MBL expression from lower primates to man, including silencing of the MBL1P1 gene and the generation of MBL2 variant structural alleles and promoter polymorphisms leading to the present human MBL2 haplotypes. We present data showing that the MBL1P1 gene has been repeatedly hit throughout evolution and silenced eventually by mutations in the glycine residues of the collagen-like region. Our results indicate that the MBL1P1 gene has been selectively turned off during evolution through the same molecular mechanisms causing the MBL2 variant alleles in man, suggesting an evolutionary selection for low-producing MBL genes.

Alleles↗
PubMed1975

Efficacy of modified human immune serum globulin in the treatment of experimental murine infections with seven immunotypes of Pseudomonas aeruginosa.

Modified immune serum globulin, prepared from human immune serum globulin by a nonenzymatic method, is apparently safe for intravenous administration to humans. The efficacy of the preparation was determined in experimental murine infections with seven immunotypes of Pseudomonas aeruginosa. Intravenously administered 0.85 percent NaCl, 0.3 m glycine, and 10 percent human albumin did not protect against lethal pseudomonas infection, whereas modified immune serum globulin given by the same route did protect mice. In the mouse protection test, the mean dose of the preparation that saved 50 percent of mice infected with any of eight strains of P. aeruginosa was 480 mg/kg (range, 12-2,333 mg/kg). For five strains the 50 percent effective dose was smaller than 200 mg/kg. There was no correlation between the efficacy of modified immune serum globulin in the mouse protection test and titers of antibody, as determined by bacterial agglutination. Therapy of pseudomonas infection in mice with modified immune serum globulin was followed by a prompt and persistent decrease in the numbers of intraperitoneal bacteria. This finding is consistent with the interpretation that modified immune serum globulin acts primarily as an opsonin and not as an antitoxin. Modified immune serum globulin may prove to be useful in the treatment of human infections.

Agglutination Tests↗
PubMed1988

Lectin-like attachment sites on murine pulmonary alveolar macrophages bind Aspergillus fumigatus conidia.

Murine pulmonary alveolar macrophages bound Aspergillus fumigatus conidia in vitro at 4 C and 37 C in the absence of serum or opsonins. This attachment was dependent on calcium and was sensitive to mild trypsinization and paraformaldehyde pretreatment of the macrophage membrane. Chitotriose, N-acetylglucosamine, D-mannose, alpha-methyl-mannoside, and L-fucose, but not D-galactose, were effective inhibitors of conidial binding. This pattern of reduction of conidial binding was consistent with that for the mannosylfucosyl receptor. In addition, conidial binding may be mediated by another lectin on the macrophage membrane, one that recognizes chitin components, because N-acetylglucosamine and chitotriose exhibited greater inhibition than expected for the mannosyl-fucosyl lectin.

Animals↗
PubMed1994

Analysis of circulating phagocyte activity measured by whole blood luminescence: correlations with clinical status.

The present study was undertaken to determine if measurement and analysis of phagocyte function are useful for diagnosis and staging of infection. Circulating phagocyte activity was measured in healthy volunteers and sequentially in patients with acute infections of different types and severity, including those with diabetes mellitus or human immunodeficiency virus (HIV) infection. Using an automated luminescence system, these phagocyte functions were measured in whole blood: basal and phorbol 12-myristate 13-acetate (PMA)-stimulated oxidase activity, basal and PMA-stimulated simple dioxygenation (e.g., oxidase-driven haloperoxidase activity), and circulating and primed opsonin receptor-dependent dioxygenation. Multiple discriminant analysis of these data showed significant differences between healthy controls, diabetic patients, HIV-positive subjects, and patients with pneumonia or sepsis syndromes. Longitudinally, circulating phagocyte function correlated with clinical condition, severity of infection, and outcome. This methodology provides rapid, objective, and sensitive diagnostic and monitoring information for patients with infections.

Acquired Immunodeficiency Syndrome↗
PubMed1996

Delta APACHE II for predicting course and outcome of nosocomial Staphylococcus aureus bacteremia and its relation to host defense.

To predict at an early phase the clinical course and outcome of nosocomial Staphylococcus aureus bacteremia, the APACHE II score at onset of bacteremia was calculated in 99 patients. A delta APACHE II score (i.e., the difference between this score and one calculated for the day before the bacteremia) was also determined. This delta APACHE II score was highly significantly correlated with clinical course (P<.001) and outcome (P<.001). The risk of a complicated clinical course and of dying from S. aureus bacteremia is determined at the very onset of bacteremia, and this risk can best be assessed by calculating the delta APACHE II score. Furthermore, a positive correlation was found between delta APACHE II scores and the level of serum opsonic activity (P=.003) toward S. aureus. Therefore, the complicated clinical course of S. aureus bacteremia is not due to a relative lack of specific opsonins.

APACHE↗
PubMed1983

Interactions of Bacteroides fragilis and phagocytes: studies with whole organisms, purified capsular polysaccharide and clindamycin-treated bacteria.

Bacteroides fragilis plays a key role in the pathogenesis of anaerobic infections and is often found mixed with aerobic organisms. We explored the interactions of this organism with phagocytes in an attempt to discern additional information about its virulence factors. We confirm an earlier report that killing of aerobic organisms by polymorphonuclear leukocytes (PMN) is decreased in the presence of high numbers of Bact. fragilis but this effect could also be demonstrated with Bact. distasonis or Staphylococcus aureus. Our data support the concept that this phenomenon may be due to competition for opsonins. Virulence of Bact. fragilis has been associated with a polysaccharide capsule. We were unable to demonstrate any deleterious effect of the purified capsular polysaccharide of Bact. fragilis on phagocytosis, killing, or chemotaxis by PMN. We were not able to demonstrate any effect of subinhibitory levels of clindamycin on the interactions of neutrophils and Bact. fragilis.

Bacteroides↗
PubMed1988

Enhanced susceptibility of gram-negative bacteria to phagocytic killing by human polymorphonuclear leucocytes after brief exposure to aztreonam.

Brief exposure of Escherichia coli, Serratia marcescens, Klebsiella pneumoniae and Salmonella typhimurium to supra-inhibitory concentrations of aztreonam enhanced their susceptibility to phagocytic killing by human polymorphonuclear leucocytes. The effect was independent of the continuous presence of the antibiotic and required the presence of serum opsonins. Phagocytic killing of Pseudomonas aeruginosa was enhanced by prior exposure to subminimal inhibitory concentrations, and killing, relative to control bacteria, was not increased with increasing concentrations of aztreonam above minimal inhibitory concentrations. The degree of sensitization, and the range of bacteria susceptible to antibiotic modulation varied between antibiotics. Under the conditions of these experiments, gentamicin sensitized pseudomonas, but failed to sensitize E. coli, while cefotaxime failed to sensitize serratia, and varied in it's activity against E. coli and pseudomonas. Enhanced killing of aztreonam-pretreated bacteria was associated with an increase in uptake by leucocytes. Aztreonam exposure decreased the liability of the bacteria to hydrophilic interactions in an aqueous two-phase partitioning system. These observations indicate that exposure of Gram-negative bacteria to aztreonam enhances phagocytic killing through modification of cell surface structures. This may be mediated through an increase in surface hydrophobicity which enhances bacterial association with leucocyte membranes with subsequent phagocytosis and intracellular killing.

Aztreonam↗
PubMed1989

The influence of cefoperazone, cefotaxime, ceftazidime and aztreonam on phagocytosis by human neutrophils in vitro.

The uptake and intracellular breakdown of Escherichia coli by human neutrophils was studied under the influence of cefoperazone, cefotaxime, ceftazidime and aztreonam. Non-opsonized as well as serum- and IgG-opsonized bacteria were used, and bacteria and neutrophils were exposed both separately before phagocytosis and together during uptake and in the postingestion phase. Preincubation of the neutrophils with the drugs stimulated their subsequent uptake of untreated serum-opsonized and non-opsonized E. coli in an antibiotic-free medium. With serum-opsonized bacteria this stimulating effect was reversed when the drugs were also present during phagocytosis. The uptake of IgG-opsonized bacteria was largely unaffected both after preincubation of the neutrophils and when the antibiotics were present during phagocytosis. With all four drugs, pre-exposure of bacteria before IgG-opsonization gave a much higher uptake than exposure after IgG-opsonization. Contrary to this, preincubation of the bacteria followed by serum-opsonization inhibited (cefotaxime, ceftazidime) or did not influence (cefoperazone, aztreonam) subsequent uptake in the absence of the drugs. The release of bacterial breakdown products from the cells in the postingestion phase was also inhibited by cefotaxime and ceftazidime after serum-opsonization. With non-opsonized and IgG-opsonized bacteria, however, a stimulation of intracellular breakdown was induced by the presence of these drugs during phagocytosis. Aztreonam and cefoperazone did not significantly influence this phase of phagocytosis. These observations suggest that the drugs bind to both neutrophils and bacteria in an individual manner and modify the binding and function of opsonins.

Anti-Bacterial Agents↗
PubMed1987

The effect of vitamin C nutriture on complement component C1q concentrations in guinea pig plasma.

This study shows that guinea pigs fed 100 times the amount of vitamin C needed for growth and for prevention of scurvy have elevated levels of complement component C1q. C1q is a plasma protein rich in hydroxyproline, an amino acid whose biosynthesis requires ascorbate. C1q is essential for host defense against pathogens, both as a component of the classical complement pathway and as an opsonin in the phagocytosis process. We measured C1q in vitamin C-depleted guinea pigs that had been repleted for 4 wks with the following daily doses of ascorbate (mg/100 g body wt): 0.50 (suboptimal), 2.0 (adequate), 10 (ample) and 50 (tissue saturating). We measured C1q in three ways: indirectly by quantifying protein-bound hydroxyproline and directly by hemolytic assay and by immunodiffusion against anti-C1q. Regardless of the method, plasma C1q was 30-50% higher in animals fed tissue-saturating ascorbate than in those fed adequate or suboptimal amounts of the vitamin (p less than 0.05, one-way analysis of variance, least significant difference test). These data confirm and significantly extend earlier work that provided indirect evidence for a relationship between C1q and ascorbate nutriture in the guinea pig. They are consistent with a possible relationship between ascorbate nutriture and host defense.

Animals↗
PubMed1989

Phagocyte metabolic functions in iron deficiency anaemia of Indian children.

Nitroblue tetrazolium test (NBT) and bactericidal activity of polymorphonuclear leucocytes was studied in 40 patients with iron deficiency anaemia (aged 0-12 years). NBT test had a significant correlation with serum iron (P less than 0.001) in all cases of iron deficiency anemia. Haemoglobin levels less than 4 g/dl also correlated significantly with NBT score. Bactericidal activity with opsonin-coated Staphylococcus aureus was considerably decreased in severe anaemia, but no significant correlation was found with serum iron concentration (P less than 0.1). The phagocytic and bactericidal activity of polymorphonuclear leucocytes is dependent upon serum iron levels and is considerably reduced in iron deficient state in anaemic children.

Anemia, Hypochromic↗
PubMed1982

The importance of splenic blood flow in clearing pneumococcal organisms.

Overwhelming infection from encapsulated bacteria occurs after splenectomy. Decreases in IgM, tufsin, and serum opsonin are known to occur in animals and humans after splenectomy. A substantial immunologic advantage exists if some splenic tissue remains, but this may not offer sufficient protection from encapsulated bacteria if splenic arterial blood flow is reduced. This experiment was designed to examine the rate of pneumococcal clearance by the spleen and to determine the relationship between splenic blood flow and splenic tissue mass in bacterial clearance from the blood. Pneumococcal clearance, splenic blood flow, and residual splenic weight were measured in 171 rabbits with normal spleens, ligated splenic arteries, splenic autotransplants, hemisplenectomies, and splenectomies. Interruption of the splenic artery results in delayed pneumococcal clearance that is due to reduced blood flow and not to a decrease in splenic tissue mass. Splenic artery ligation to preserve an injured spleen cannot be assumed to give protection from sepsis.

Animals↗
PubMed1988

The effects of complement activation during cardiopulmonary bypass. Attenuation by hypothermia, heparin, and hemodilution.

Complement activation was examined prospectively in 100 cardiopulmonary bypass (CPB) patients. Plasma C3a desArg (C3a) increased (cannulation: 234 +/- 33 ng/mL; 20 minutes on CPB: 622 +/- 51; 2 hours after CPB: 1143 +/- 109, p less than 0.0001). C3a at 2 hours was higher in the 13 patients requiring mechanical ventilation for longer than 1 day (1023 +/- 274) than in the 67 without respiratory complication (568 +/- 45, p less than 0.004). Five more patients were studied for neutrophil activation to confirm that a biologic effect of complement activation occurs during CPB; in these five patients C3a increased to 317% of baseline after 10 minutes on CPB with a corresponding rise in neutrophil cell surface receptors for the complement opsonin C3b (as measured by indirect immunofluorescence) to 168% (p less than 0.05). Both increases were sustained at 30 minutes. Temperature, dilution, and heparin were studied as variables relevant to CPB. Exposure of normal neutrophils to C5a in vitro caused an increase in C3b receptors which was dependent on temperature (0 specific fluorescence at 0 C, 30 at 25 C, 180 at 30 C, and 275 at 37 C). Generation of C3a and C5a in normal serum by zymosan was also temperature-dependent (ng/mL C5a generated: 0.7 at 25 C, 200 at 30 C, and 897 at 37 C; ng/mL C3a generated: 546 at 25 C, 10,872 at 30 C, and 65,667 at 37 C). Serum dilution to 33% decreased ng/mL C5a generated in the same system from 200 to 76 with no effect on C3a. Addition of heparin to 20 U/mL decreased ng/mL C3a generated from 10,872 to 913 and C5a from 200 to 8. Thus, hypothermia, dilution, and heparin protect CPB patients from complement activation by reducing both generation of C3a/C5a and the subsequent cellular response of neutrophil activation.

Cardiopulmonary Bypass↗
PubMed1987

Cryoprecipitate infusion fails to improve organ function in septic shock.

Plasma fibronectin may be of critical importance for the septic patient through its proposed function as the major opsonin for macrophage clearance of circulating, noncellular debris. As a rule, critically ill, septic patients are depleted of fibronectin. In earlier uncontrolled studies, infusion of fibronectin-rich cryoprecipitate had resulted in improved renal and pulmonary functions and changes in peripheral hemodynamics. In this controlled study, 32 septic ICU patients (mean initial fibronectin level = 60% of normal) received cryoprecipitate or control infusions. Although the fibronectin level was significantly elevated to the normal range in the cryoprecipitate group, no effects were seen in hemodynamics, oxygen metabolism, or lung and kidney functions. Our results indicate that this form of fibronectin therapy does not influence the impaired organ function in septic shock.

Adult↗
PubMed1975

Changes in the serum alpha glycoprotein distribution in trauma patients.

Serum electropherograms of trauma patients, when stained for glycoproteins, show striking changes in the alpha glycoproteins of these patients which revert to normal if and when they recover. Thus monitoring of the glycoprotidograms of trauma patients (contrary to ordinary protidograms) is likely to afford important information on the course of these patients' recovery. Of two glycoproteins that have been more closely studied, one, alpha1A acid glycoprotein, is a phagocytosis inhibitor; it is increased in the sera of trauma patients. The other, alpha2HS glycoprotein, is a phagocytosis promotor (or opsonin); it is decreased in the sera of trauma patients. Both the increase of the first and the decrease of the second glycoprotein may thus contribute to the known increased proneness to bacterial infection among these patients.

Electrophoresis, Cellulose Acetate↗