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PubMed1995

Mannan-binding protein and complement dependent opsonization in alcoholic cirrhosis.

Mannan-binding protein is synthesized by the liver and functions in first-line host defence by opsonizing mannose-rich microorganisms due to activation of the classical complement pathway independent of Clq, and by an intrinsic ability to opsonize and mediate phagocytosis. We have investigated whether the increased susceptibility to bacterial infections in patients with cirrhosis could be explained by low plasma concentrations of mannan-binding protein and impaired complement-dependent opsonization. We examined 51 patients with compensated alcoholic cirrhosis, 34 who were decompensated and 16 healthy controls. Irrespective of group, we found a significant correlation (p < 0.05) between plasma mannan-binding protein concentration and deposition of the complement opsonin C4 on mannan from baker's yeast. In contrast to what was expected, this kind of opsonization and plasma levels of mannan-binding protein were significantly increased in the patients with decompensated cirrhosis (p = 0.01 and p = 0.007, respectively). A significant correlation (0 < 0.05) was found between mannan-binding protein and erythrocyte sedimentation rate, fibrinogen and haptoglobin in these patients. Though the correlations were weak (rho = 0.49, rho = 0.48 and rho = 0.40, respectively), the elevated levels of mannan-binding protein in the patients with decompensated cirrhosis may reflect an acute phase reaction. It is concluded that plasma levels of mannan-binding protein are increased in patients with decompensated cirrhosis and that complement-dependent opsonization of mannan does not seem to be compromized in patients with alcoholic cirrhosis.

Adult↗

PubMed1993

Eubacterium yurii subspecies margaretiae is resistant to nonopsonic phagocytic ingestion.

We have previously shown that strains of Eubacterium yurii are hydrophobic, as compared with human polymorphonuclear leukocytes (PMNs), possibly because of a crystalline surface layer (S-layer) covering the cell envelope of this potential endo-perio pathogen. The aim of the present study was to investigate the phagocytic ingestion by PMNs of the three E. yurii subspecies, with special attention to bacterial surface structures and hydrophobicity. Type strains of subspp. margaretiae, yurii, and schtitka, together with three clinical isolates from necrotic root canals, were studied. All strains were hydrophobic when tested by a two-phase partition method. E. yurii subspp. margaretiae strains ATCC43715T, ES4C, and ES14B-8E were resistant to PMN ingestion in the absence of opsonins, whereas strains of the two other subspecies were readily ingested. The presence of a resistant strain (subsp. margaretiae ATCC43715T) did not inhibit the ingestion of a sensitive strain (subsp. schtitka ATCC43716T). Ingestion of E. yurii subsp. margaretiae strains required opsonization by normal human serum or specific antibodies. Electron microscopy revealed an S-layer in all strains and fimbria-like structures in the subspp. margaretiae and yurii strains. The antiserum prepared against the S-protein of E. yurii subsp. margaretiae ATCC43715T showed only slight cross-reactivity with other E. yurii strains and indicated the presence of strain-specific rather than species- or subspecies-specific antigens in the S-protein of E. yurii subsp. margaretiae ATCC43715T. The results suggest that the mere presence of the S-layer or fimbria-like structures cannot explain the susceptibility to ingestion by the PMNs.(ABSTRACT TRUNCATED AT 250 WORDS)

Alkanes↗

PubMed1992

Phagocytosis of Porphyromonas gingivalis and Prevotella intermedia by human polymorphonuclear leukocytes in clots formed from human plasma or purified fibrinogen.

We have studied the polymorphonuclear leukocyte (PMN) phagocytosis of Porphyromonas gingivalis (Bacteroides gingivalis) and Prevotella intermedia (Bacteroides intermedius) in three-dimensional fibrin meshworks formed by either clotting human plasma or by clotting purified fibrinogen with human serum incorporated. PMN phagocytosis of rhodamine isothiocyanate-labelled P. gingivalis in the fibrinogen clot was 43 +/- 6% and in the plasma clot was 62 +/- 10%; for P. intermedia the values were 15 +/- 10% and 63 +/- 10%, respectively. This showed that phagocytosis in the plasma clot system was significantly higher than in the fibrinogen clot system. However, increasing the concentration of serum in the fibrinogen clot abolished this difference, suggesting that opsonin levels determine PMN function in this assay. Labelling the bacteria with a different fluorochrome, fluoroscein isothiocyanate, reduced the level of PMN phagocytosis in both three-dimensional systems but phagocytosis was unaffected when PMN were adherent to glass. It would appear that PMN phagocytosis in three-dimensional systems can vary depending on the composition of the clot and the fluorochrome used to label the bacteria.

Analysis of Variance↗

PubMed1985

Opsonization of bacteria by uterine secretions of cyclic mares.

Uterine flushings collected from mares before and after bacterial-induced inflammation were assayed for ability to opsonize Streptococcus zooepidemicus for phagocytosis by polymorphonuclear leukocytes. Opsonization was measured as the peak phagocytic rate of bacteria preincubated with uterine flushings relative to the peak phagocytic rate of unopsonized bacteria. Flushings from four mares with noninfected uteri were unable to opsonize bacteria regardless of whether uteri were flushed at estrus or on day 10 postovulation. In a second experiment, 7 X 10(9) live S. zooepidemicus were inoculated into the uterus of five mares during estrus. Uterine flushings collected at the estrus before inoculation or at the estrus after inoculation did not opsonize bacteria. Four of five flushings collected 6 hr post inoculation, however, were capable of opsonization. Based on heat inactivation at 56 degrees C, the opsonizing activity of one of four flushes was due to a complement protein. It was concluded that one aspect of the acute inflammatory response of the mare's uterus is accumulation of opsonins in the uterine lumen.

Animals↗

PubMed1978

Cryoprecipitate reversal of opsonic alpha2-surface binding glycoprotein deficiency in septic surgical and trauma patients.

Human opsonic alpha2-surface binding glyoprotein (alphs2SB-glycoprotein), a molecule having immunologic identity with an amino acid composition similar to cold-insoluble globulin, is concentrated in a cryoprecipitate of plasma. Septic surgical and trauma patients manifesting opsonic alpha2SB-glycoprotein deficiency and associated reticuloendothelial system dysfunction were treated by intravenous infusion of cryoprecipitate. This therapy restored circulating bioreactive and immunoreactive opsonin and improved their septicemia, pulmonary insufficiency, and duration of recovery. Cryoprecipitate infusion may offer a new approach to the treatment of septic injured patients in preventing multiple organ failure; measurement of immuno-reactive serum opsonic alpha2SB-glycoprotein may provide a noninvasive index of reticuloendothelial system function and patient status during servere sepsis that follows trauma.

Cryoglobulins↗

PubMed1975

Superficial antigens of Campylobacter (Vibrio) fetus: characterization of antiphagocytic component.

A glycoprotein responsible for the antiphagocytic properties of Campylobacter fetus has been identified by comparing cells of a wild-type strain with those of a mutant lacking this substance. The antiphagocytic component is demonstrable through electron microscopy as a discrete, negatively charged structure on the periphery of the cell. CLT is readily removed from the cell by mild extraction procedures and contributes to the inagglutinability in O antiserum normally displayed by C. fetus. Cells possessing this antigen are refractory to ingestion by macrophages except in the presence of specific antiserum. In its absence maximum phagocytosis occurs without a requirement for opsonins. It is concluded that the antiphagocytic component comprises a critical virulence factor of the bacterium.

Animals↗

PubMed1975

Biochemical and immunological properties of ribonucleic acid-rich extracts from Francisella tularensis.

Ribonucleic acid (RNA)-rich extracts derived from the attenuated strain of Francisella tularensis (strain LVS) protected Swiss mice against lethal challenge with F. tularensis strain 425 but not against strain SCHU S4. No killed preparation, including an RNA-rich extract from SCHU S4 itself, offered protection against strain SCHU S4 in contrast to the high level of protection offered against this strain by vaccination with live strain LVS. The protective activity observed against strain 425 was sensitive to ribonuclease but not to Pronase. Protective activity is not a general property of bacterial RNA, since RNA-rich extracts from Staphylococcus aureus offered no protection against tularemia, although disc gel electrophoresis showed similar kinds and amounts of RNA in preparations form F. tularensis and S. aureus. Furthermore, inability to localize activity to a specific region in sucrose gradients suggests a structural rather than an informational role for the RNA in such extracts. RNA-rich extracts from F. tularensis but not from S. aureus were efficient inducers of F. tularensis opsonins in mouse serum, suggesting one mechanism by which such extracts confer protection.

Animals↗

PubMed1977

Evaluation of serum opsonic capacity by quantitating the initial chemiluminescent response from phagocytizing polymorphonuclear leukocytes.

Phagocytically activated polymorphonuclear leukocytes produced a chemiluminescence that could be correlated metabolically with the stimulated oxidation of glucose via the hexose monophosphate shunt, The chemiluminescence observed was considered to originate from the relaxation of electronically excited carbonyl groups produced during singlet molecular oxygen-mediated microbicidal oxidation of the ingested microbe. With adequate adjustment of leukocyte and bacterial concentrations, the rate of chemiluminescence increase was nearly constant for the first minutes after initiation of phagocytosis. This rate was dependent on the quantity of bacteria phagocytized by the leukocytes. If both leukocytes and bacterial concentrations were held constant, this initial rate of chemiluminescence reflected the opsonic capacity of the sera used for opsonization. The prior absorption of opsonins from serum resulted in a decresed rate of chemiluminescence related to the quantity of bacteria used for absorption. Heating of sera to 56 degrees C for 30 min resulted in a great decrease in the chemiluminescent responses and may reflect the role of complement in the opsonization process.

Adult↗

PubMed1977

Kinetics of phagocytosis and intracellular killing of Candida albicans by human granulocytes and monocytes.

The study of the phagocytosis and intracellular killing of Candida albicans by granulocytes and monocytes has been hampered by the budding and pseudomycelium formation of this yeast during a relatively short incubation period at 37 degrees C and by the similar density of candida cells and phagocytes, which makes differential centrifugation impossible. In the present study, C. albicans was used after 5 days of preculture at 30 degrees C, after which the number of candida cells remained constant during incubation at 37 degrees C for 90 min. On this basis, phagocytosis and intracellular killing were limited to a period of 60 min. Phagocytosis of C. albicans by granulocytes and monocytes was measured with a hemocytometer, the number of extracellular candida being a measure of the ingestion of these microorganisms. After 60 min, 96% of the candida cells were ingested by normal human granulocytes and monocytes. This process was dependent on the opsonin concentration and temperature and was inhibited by mono-iodoacetic acid. Heat-inactivated serum was less active than fresh serum, reflecting the role of complement factors with respect to opsonization. Intracellular killing was measured by a microbiological assay. After 60 min of incubation of phagocytes together with C. albicans and serum, human granulocytes and monocytes killed 58 and 50% of the ingested candida, respectively. This process was inhibited by phenylbutazone. Phagocytes from patients with chronic granulomatous disease showed impaired intracellular killing.

Candida albicans↗

PubMed1978

Role of antibody and complement in opsonization of group B streptococci.

A requirement for the classic complement pathway in opsonization of group B streptococci was observed by using both a chemiluminescence and a radiolabeled bacterial uptake technique. The classic pathway increased levels of opsonization for types Ia and II stock and wild strains and for some type III wild strains. In contrast, other type III wild strains and the type III stock strain had accelerated kinetics of uptake in the presence of an intact classic pathway, but the level of opsonization was unchanged from that with antibody alone. We could not demonstrate a significant role for the alternative pathway in opsonizing stock or wild strains of group B streptococci. Futhermore, electrophoretic and complement consumption analysis by hemolytic titration failed to reveal alternative pathway activation by the majority of strains of this group. Therapy aimed at supplying opsonins for these organisms will require the presence of type-specific antibody.

Antibodies, Bacterial↗

PubMed1978

Structural analysis of the cellular constituents of a fresh clinical isolate of Staphylococcus aureus, and their role in the interaction between the organisms and polymorphonuclear leukocytes in the presence of serum factors.

The in vitro interaction of a fresh clinical isolate of Staphylococcus aureus and polymorphonuclear leukocytes was investigated. The importance of the various cellular constituents as host immunological factors was analyzed, and the results suggested that two components, namely, an acidic polysaccharide consisting of a predominance of aminogalacturonic acid and a strain-specific mucopeptide complex, may be involved in impeding in vitro opsonization of the organism by leukocytes. Immunochemical analysis indicated that the acidic polysaccharide possessed the same immunodominant aminogalacturonic acid residues as the antiphagocytic acidic antigen of the encapsulated prototype Scott strain. Antisera derived from rabbits immunized with strain D contained two types of opsonins, those with acidic polymer specificity and those with mucopeptide complex specificity.

Antigens, Bacterial↗

PubMed1978

Immunity to antigenically related salmonellae: effects of humoral factors on the bactericidal activity of normal and immune peritoneal exudate cells.

Immunity against Salmonella enteritidis and Listeria monocytogenes was studied by measuring in vitro the bactericidal activity of peritoneal exudate cells (PEC) of control (normal PEC) and S. typhi Ty2-immune (immune PEC) mice. Specific immune serum, anti-S. tyhphi Ty2, heat inactivated at 56 degrees C for 30 min, significantly inhibited the growth of S. enteritidis only with immune PEC. These opsonic factors had no effect upon the activity of normal PEC. That such inhibition could not be demonstrated in Listeria experiments, either with immune or normal PEC, suggests that S. enteritidis was specifically recognized, in vitro, by the thermostable opsonin anti-S. typhi Ty2 and that macrophages from immune PEC were more efficient in inhibiting bacterial growth than those from normal PEC. Thus, the interaction between macrophages and the microorganism seems to play an essential role in cell-mediated as well as humoral immunity.

Antibodies, Bacterial↗

PubMed1979

Effect of pili on susceptibility of Escherichia coli to phagocytosis.

The degree of piliation of 20 clinical isolates of Escherichia coli was correlated with their susceptibility to phagocytosis by human polymorphonuclear leukocytes. Piliation was quantitated by negative staining, and phagocytosis was quantitated by a monolayer technique. Ingestion was confirmed by electron microscopy. In the absence of source of opsonins, there was a positive correlation between the degree of piliation and susceptibility to phagocytosis (y = 0.83x + 19.58; correlation coefficient = 0.65; P < 0.01). Heavily piliated strains were no longer phagocytized after their pili were removed by ultraviolet irradiation. Phagocytosis was reduced 75% in the presence of 0.1 M d-mannose, an agent which competitively inhibits binding of pili to cell surfaces. l-Mannose, d-glucose, and d-galactose were much less inhibitory. The viability of piliated organisms was reduced by 1 log after 1 h of incubation with polymorphonuclear leukocytes. Addition of 10% fresh human serum increased both the rate and completeness of killing. These observations suggest that polymorphonuclear leukocytes may interact with the pili of E. coli to promote phagocytosis. This phenomenon may have clinical relevance in situations where normal opsonic activity is poor, such as the renal medulla.

Binding Sites↗

PubMed1980

Cytophilic and opsonic antibodies in visceral leishmaniasis in mice.

Although acquired immunity to leishmaniasis is generally considered to be cell mediated, humoral factors may be partially responsible. The present study showed that antisera from C57BL/6J mice superinfected with Leishmania donovani contained cytophilic antibody and opsonins for both the amastigote and promastigote stages of the parasite. Macrophages treated with mouse hyperimmune serum in an in vitro macrophage culture system bound statistically significantly more parasites at 4 degrees C (and subsequently phagocytized them at 37 degrees C) than did macrophage cultures treated with control serum. The percentages of antibody-treated macrophages bearing and containing parasites were also significantly greater than the percentages of control serum-treated macrophages bearing and containing parasites, respectively. These differences persisted in cultures during a 9-day observation period when sera from mice killed 10 or 11 days after superinfection were used. However, when sera from mice killed 24 days after superinfection were tested with amastigotes, by day 9 the number of parasites and the percentage of cells parasitized in the culture decreased to control values or significantly below them. Thioglycolate-stimulated macrophages treated with hyperimmune serum bound more amastigotes at 4 degrees C than did stimulated macrophages treated with control serum. Activated macrophages also demonstrated increased nonspecific binding of amastigotes. Treatment of macrophages with trypsin reduced both cytophilic antibody-specific and nonspecific binding of amastigotes. The demonstration of in vitro effects of anti-leishmanial antibody from superinfected mice might indicate a possible role for humoral antibody in immunity to leishmaniasis in mice.

Animals↗

PubMed1981

Participation of immunoglobulins and complement components in the intracellular killing of Staphylococcus aureus and Escherichia coli by human granulocytes.

Immunoglobulins and complement components are required for optimal ingestion and optimal killing of microorganisms by granulocytes. The degree of opsonization of microorganisms necessary for their ingestion was lower than that required for the killing of these bacteria during the ingestion phase. Killing during this phase was found to depend mainly on the presence of heat-labile opsonins, probably C3b, present on the microorganisms. Extracellular immunoglobulin G (IgG) and C3b were indispensable for optimal intracellular killing after ingestion was complete. This was established with an assay permitting assessment of the course of the number of viable intracellular bacteria independent of the ingestion of new live bacteria. Maximal intracellular killing by human granulocytes of ingested catalase-positive (Staphylococcus aureus and Escherichia coli) or catalase-negative (Streptococcus pyogenes and S. pneumoniae) microorganisms was found only when fresh serum was present extracellularly. Killing was suboptimal in the absence of serum. With heat-inactivated serum, the killing index lay between the indices obtained in the presence and absence of fresh serum. The stimulatory activity of heat-inactivated serum was most probably due to the interaction of IgG with the Fc receptor on the granulocyte membrane, since IgG subclasses IgG1 and IgG3 as well as pFc fragments of IgG stimulated the intracellular killing to the same degree as heat-inactivated serum did. In addition, (Fab1)2 fragments of IgG did not stimulate killing, and reduced killing was observed in the presence of heat-inactivated serum after reduction of the number of Fc receptors. The extra stimulation of the killing process in the presence of fresh serum compared with heat-inactivated serum was due to the interaction between membrane receptors and complement--most probably C3b generated by both the classical and the alternative pathways of complement activation. This conclusion is based on results obtained with sera in which one or both complement pathways were blocked, on the restoration of the killing-stimulatory activity of C3-deficient serum after addition of fresh C3, and on the reduced killing observed in the presence of fresh serum after reduction of the number of C3 receptors by the use of pronase or antigranulocyte serum.

Blood Bactericidal Activity↗

PubMed1981

Human fibronectin binding to staphylococcal surface protein and its relative inefficiency in promoting phagocytosis by human polymorphonuclear leukocytes, monocytes, and alveolar macrophages.

The interaction between human fibronectin and 17 strains of staphylococci was studied in an attempt to elucidate the staphylococcal cell wall component(s) involved in fibronectin binding and to determine the influence of fibronectin upon phagocytosis by three types of phagocytic cells. Purified, radiolabeled fibronectin bound to a similar degree to six laboratory strains and three fresh clinical isolates of Staphylococcus aureus; similar binding of fibronectin was found with S. aureus strains deficient in cell wall teichoic acid or clumping factor and coagulase, as well as with three strains of S. epidermidis. There was minimal binding of fibronectin to encapsulated S. aureus and to Escherichia coli. Fibronectin bound to intact cells and to a crude cell wall preparation of S. aureus H, but not to purified cell walls or peptidoglycan. Trypsinization of staphylococci prevented subsequent fibronectin binding, but binding did not correlate well with the protein A content in S. aureus cell walls. At physiological concentrations, fibronectin binding to staphylococci did not promote phagocytosis of bacteria by human polymorphonuclear leukocytes, monocytes, or alveolar macrophages. Also, depletion of fibronectin from normal human serum did not result in a measurable loss of opsonic activity for staphylococci. It is concluded that fibronectin binding to staphylococci involves a surface protein shared among strains of S. aureus and S. epidermidis, and that in comparison to C3b and IgG, fibronectin plays a relatively minor role as an opsonin for staphylococci.

Bacterial Proteins↗

PubMed1982

Serotype-specific opsonization of Treponema hyodysenteriae.

Treponema hyodysenteriae was shown to attach to mouse peritoneal cells in the absence of serum opsonins in vitro. If serotype-specific antiserum from pigs was added to the media and treponemes of that corresponding serotype were employed in the assay, the amount of attachment increased an average of 3.7 times that of the control without pig sera. However, the amount of attachment was increased an average of only 1.5 times that of the control if organisms of any noncorresponding serotype of T. hyodysenteriae were used in the assay. Since the lipopolysaccharide (LPS) extracted from T. hyodysenteriae is the basis for serotyping the treponeme, the ability of these distinct LPS types to block attachment by blocking opsonization of the organisms was tested. Attachment, using corresponding antisera and treponemes, was blocked by LPS extracted from treponemes of that serotype, but not by LPS extracted from treponemes of other serotypes. These results indicate that antibody response to T. hyodysenteriae infection in pigs is serotype-specific. Furthermore, since opsonization and subsequent attachment of bacteria to phagocytes is known to be a protective mechanism, we suggest that the LPS may be an important antigen in the stimulation of host defense against the treponeme.

Animals↗

PubMed1983

Human peritoneal macrophage phagocytic, killing, and chemiluminescent responses to opsonized Listeria monocytogenes.

Opsonization with normal human serum, purified immunoglobulin G, or immunoglobulin G-deficient serum promoted phagocytosis of Listeria monocytogenes by human peritoneal macrophages. However, normal human serum was the most effective opsonin in elicting killing and chemiluminescent responses. Macrophages phagocytized and killed almost as much as polymorphonuclear leukocytes but produced considerably less chemiluminescence.

Ascitic Fluid↗