Influence of preformed antibody on the pathogenesis of experimental Candida albicans endocarditis.

The influence of preformed antibody on the induction of experimental Candida albicans endocarditis was investigated by both in vitro and in vivo techniques. Preincubation of C. albicans with immune serum (raised in rabbits by intravenous injection of Formalin-killed yeast cells) decreased adhesion to the constituents of nonbacterial thrombotic endocarditis, e.g., fibrin plus platelets, in vitro. Two different methods, with radiolabeled or viable yeast cells, were confirmatory and demonstrated decreased adhesion of immune serum-treated C. albicans cells to 0 to 7.8% of control values (P less than 0.001). These results correlated with protection from the development of C. albicans endocarditis in the immunized rabbits. The mean (+/- standard deviation) infectious dose for 50% of the animals was 10(5.29) +/- 10(0.07) in 48 control animals versus 10(7.11) +/- 10(0.22) in 37 immunized rabbits (P less than 0.001). These studies suggest that humoral antibody may protect against C. albicans endocarditis, perhaps through inhibition of adhesion, a crucial early step in the pathogenesis of endocarditis.     

Effect of immunization on the genesis of pneumococcal endocarditis in rabbits.

The effect of immunization with whole organisms on the development of Streptococcus pneumoniae endocarditis was examined by in vivo and in vitro methods. Immunization protected rabbits from pneumococcal endocarditis when the in vivo catheterization model was used. The inoculum size that caused endocarditis in 50% of the unimmunized rabbits was 1.1 X 10(5) colony-forming units, whereas 1.2 X 10(7) colony-forming units were required for infecting 50% of the immunized rabbits (P less than 0.001). Investigations were carried out to determine the mechanism which enabled immunization to prevent the development of pneumococcal endocarditis; they indicated that a reduction in bacterial adherence could not explain this phenomenon. In vitro studies showed that subagglutinating quantities of antibody increased the adherence (P less than 0.05) of pneumococci to rabbit aortic valve cusps. The adherence ratio of pneumococci to fibrin-platelet clots was at least doubled by the presence of subagglutinating dilutions of immune sera (P less than 0.001). Further studies showed that immunoglobulin G in the immune sera accounted for this increased in vitro adherence. However, further in vivo studies showed that immunized rabbits were able to clear live pneumococci from their bloodstreams within 4.5 h, whereas unimmunized rabbits failed to clear the organism within 24 h.     

Influence of Staphylococcus aureus antibody on experimental endocarditis in rabbits.

To evaluate the potential protective benefit of antibody to whole cells of Staphylococcal aureus for the prevention of endocarditis, the rabbit endocarditis model was used. Methicillin-sensitive (17A) and methicillin-resistant (173) S. aureus strains were evaluated in rabbits with or without indwelling intracardiac catheters. All immunized rabbits developed significant homologous agglutinating antibody titers (the mean reciprocal titers were 15,300 to strain 17A and 1,150 to strain 173). After challenge, virtually no significant differences were observed between immunized and unimmunized animals with respect to (i) incidence of endocarditis, (ii) concentration of bacteria in infected vegetations, (iii) incidence of metastatic renal abscesses, or (iv) concentrations of bacteria in infected kidneys. The clearance of homologous S. aureus strains from blood cultures was similar for immunized and unimmunized animals at 10 to 90 min after intravenous challenge. In vivo adherence of homologous S. aureus strains to aortic valves and vegetations was similar in immunized and unimmunized animals when evaluated at 30 and 90 min postchallenge. Even without catheterization, the incidence of bacteremia and renal abscesses was the same in immunized and unimmunized rabbits. Whole-cell-induced S. aureus antibody did not prevent or modify any stage in the development of endocarditis in rabbits.     

Adherence of Streptococcus sanguis to conformationally specific determinants in fibronectin.

The adherence of Streptococcus sanguis to specific receptors exposed or deposited at the site of endothelial damage may play an important role in the development of infective endocarditis. Adherence of the Challis strain of S. sanguis to gelatin (or collagen) and gelatin-binding components of plasma was examined with an enzyme-linked immunosorbent assay. S. sanguis adhered poorly to immobilized gelatin and to molecular or fibrillar collagen. However, in the presence of fresh human plasma, the adherence of S. sanguis to all three substrates increased as much as eightfold. Removal of gelatin-binding proteins eliminates the ability of plasma to enhance adherence of S. sanguis to the substrates. Addition of purified human plasma fibronectin (Fn) to the absorbed plasma restored the adherence-promoting ability in a dose-dependent manner. A similar dose-dependent increase in S. sanguis adherence was observed when increasing concentrations of Fn alone were added to the gelatin-coated assay wells. S. sanguis adherence to immobilized fibronectin could not be inhibited by preincubating either the bacteria or the gelatin-coated assay wells with Fn or by including excess soluble Fn in the assay mixture. Studies with peptides purified from trypsin digests of Fn indicated that the 160- to 180-kilodalton (kDa) fragments which retain both the gelatin-binding and the cell-binding regions of the intact molecule support adherence of S. sanguis to gelatin. The 160- to 180-kDa fragments inhibited the interaction of S. sanguis with immobilized Fn. In contrast, intact Fn and the 31-kDa amino-terminal fragment were unable to inhibit the adherence when used in equivalent or greater molar amounts. These in vitro results suggest that in the presence of whole plasma, S. sanguis binds to immobilized gelatin or collagen via Fn bound to the immobilized substrates. Our finding that adherence of S. sanguis to immobilized Fn can occur in the presence of large concentrations of Fn, whether in plasma or purified, indicates that a S. sanguis-binding domain is cryptic in the Fn molecule while in solution and is exposed by a conformational change when the Fn becomes bound to gelatin-coated plastic. The ability of peptide fragments of Fn to inhibit S. sanguis adherence is consistent with this hypothesis.     

Experimental Candida albicans endocarditis: characterization of the disease and response to therapy.

Endocarditis caused by Candida albicans was induced in rabbits after insertion of a catheter across the aortic valve. The mean survival time of 34 rabbits was 26 days. Only 7% of temperature recordings taken were elevated. Candida was recovered from only 9% of blood cultures taken. Precipitating and agglutinating serum antibody was detected after 12 days of infection. Antibody titers rose progressively until death in rabbits with endocarditis, whereas titers peaked early and subsequently decreased in animals that received an intravenous injection of C. albicans without precatheterization. Three groups of rabbits were treated for 6 days with amphotericin B, 5-fluorocytosine, or the two durgs in combination. Amphotericin B alone reduced the mean titer of organisms from log10 8.79 +/- 1.46 to log 10 3.1 +/- 1.9 colony-forming units/g. 5-Fluorocytosine was less effective (mean titer after 6 days of therapy was log10 7.4 +/- 0.33 colony-forming units/g). The addition of 5-fluorocytosine to amphotericin B did not increase the rate at which Candida cells were eradicated from the vegetations. These in vivo results corrleated with the failure to demonstrate an increased rate of fungicidal activity in vitro with the two drugs.     

Effects of molecular weight of dextran on the adherence of Streptococcus sanguis to damaged heart valves.

Dextran-producing streptococci such as Streptococcus sanguis are the organisms most frequently associated with infective endocarditis in humans. A series of experiments was designed to study how the molecular weight of dextrans affects the adherence of an endocarditis strain of S. sanguis to canine heart valves covered with platelets and fibrin. The data indicated that this adherence was dependent on dextrans of high molecular weight, such as dextran T-2000 or glucans isolated from S. sanguis or S. mutans. The adherence properties of the strain studied were not modified by prior exposure of the bacterial cells of valve leaflets to high-molecular-weight dextrans. Preexposure of bacterial cells or valve leaflets to low-molecular-weight dextrans decreased their adherence. Low-molecular-weight dextrans interfered with adherence of dextran-positive strains to damaged heart valves.     

In vitro studies of dental plaque formation: adsorption of oral streptococci to hydroxyaptite.

A mixture of saliva-coated hydroxyapatite beads and radioactively labeled bacteria has been employed as an in vitro model for the initial phase of dental plaque formation. Adsorption in this model can be expressed by the Langmuir adsorption isotherm, and the adherence of oral streptococci can be expressed as the product of the affinity constant (Ka) and the number of binding sites (N), KaN. With this approach, Streptococcus sanguis serotype 1 strains adhered better (KaN = [187 +/- 72] X 10(-2)) than serotype 2 strains (KaN = [97 +/- 84] X 10(-2)); a t test showed this difference to be statistically significant to the 99.99% confidence level. Strains of S. mitis, S. mutans, and S. salivarius did not appear to adhere as well. To analyze the bacterial receptors involved in adherence, competition studies in which increasing quantities of unlabeled bacteria were added to a fixed quantity (4 X 10(9) cells per ml) of 3H-labeled serotype 1, reference strain S. sanguis G9B, were performed. These studies indicated that the type 1 strains competed for the same, or closely related, binding sites. Competition studies using serotype 2 S. sanguis strains resulted in an increased binding of reference strain G9B to hydroxyapatite. Scanning electron microscopy indicated this effect was due to the formation of localized aggregations of bacteria, presumably representing the two bacterial types. The results of competition studies with S. mitis were variable, and several strains of other oral bacteria showed little or no competition.     

Adherence of Porphyromonas (Bacteroides) gingivalis to Streptococcus sanguis in vitro.

Intergeneric bacterial adherence is responsible for the complexity of the microbiota in human dental plaque and is believed to enable some extraneous bacteria to initially colonize the human oral cavity. Some current evidence indicates that Streptococcus sanguis, an early colonizer of teeth, enhances subsequent colonization by Porphyromonas (Bacteroides) gingivalis, a bacterium associated with advanced adult periodontitis. In this study, selected strains of P. gingivalis and S. sanguis were tested for their adherence activities in vitro. A differential filtration assay was devised in which one member of the test pair was radiolabeled. Heterogeneous aggregates that formed in mixed suspensions were collected on polycarbonate filters (8-microns pore size) and were washed free of individual bacteria and small homologous clumps. P. gingivalis 381, W50, JKG7, and 33277 adhered to S. sanguis G9B, M5, Challis 6, and 38. P. gingivalis A7A1-28 did not adhere well to S. sanguis under these conditions. More precise measurements of intergeneric adherence were obtained with an alternative assay with radiolabeled P. gingivalis and an artificial dental plaque composed of S. sanguis coupled to cyanogen bromide-activated agarose beads. CNBr-agarose was selected as the supporting matrix for the plaque because it was uniformly and permanently coated with S. sanguis and because P. gingivalis had negligible adherence activity for streptococcus-free beads. P. gingivalis W50 grown to the early stationary phase adhered to S. sanguis-coated beads in higher numbers than either midlogarithmic- or late-stationary-phase cells. Intergeneric adherence was not inhibited or reversed by the presence of lactose or other monosaccharides or disaccharides. Pretreatment of either bacterium with trypsin or proteinase K reduced subsequent adherence by 86 to 100%. Neuraminidase treatment of P. gingivalis caused 98% reduction of adherence, whereas similar treatment of S. sanguis caused only a 2% loss. Preincubation of P. gingivalis at 60 degrees C for 30 min decreased subsequent adherence to S. sanguis-coated beads by 94%. Adherence was reduced by 96% when bacteria were assayed while suspended in human whole saliva or when pretreated with saliva and subsequently assayed in buffer. The concentration of whole human saliva required to inhibit 50% adherence in this assay was 23 micrograms per ml (1:200 dilution). Suspension of the bacteria in normal rabbit serum resulted in 94% inhibition of adherence. These data indicate that saliva and serum may be important host defense factors for controlling Porphyromonas-Streptococcus adherence.     

Role of granulocytes in experimental Streptococcus sanguis endocarditis.

We investigated the role of granulocytes during the induction and course of experimental Streptococcus sanguis endocarditis in rabbits by depleting blood granulocytes with nitrogen mustard. The induction of the endocarditis was not influenced by granulocytopenia: the 50% infectious dose was 5.4 X 10(4) colony-forming units in normal and granulocytopenic rabbits. However, granulocytopenia influenced the curse of the endocarditis, as shown by a significant increase in the number of colony-forming units per gram of vegetation (P less than 0.02) from 24 to 72 h after the injection of 10(5) colony-forming units of S. sanguis. This rise did not occur in the control rabbits. Furthermore, bacteremia was significantly higher in the granulocytopenic rabbits (P less than 0.05) during the first 48 h compared with the control rabbits. This was not because of altered clearance of the streptococcus inoculum or seeding of streptococci from extracardiac bacterial foci. We concluded that granulocytes have no measurable effect on the induction of S. sanguis endocarditis, but during the course of the endocarditis, granulocytes keep the endocardial infection in check.     

Cellular and humoral autoimmune responses against human eye muscle membrane antigen in Graves' disease

75 patients with Graves' disease (54 with ophthalmopathy) were investigated using the tests of leucocyte adherence inhibition and immune adsorption with 125I-labelled Staphylococcus Protein A, against human eye muscle "crude" membrane antigen. The results of positive leucocyte adherence inhibition (10 out of 26 vs. 1 out of 28, P less than 0.05) and anti-human eye muscle membrane antibody index (mean +/- S.D.) (1.89 +/- 1.20 vs. 0.84 +/- 0.38, P less than 0.001) showed a correlation with the patients with clinically active eye disease and the HLA-B8 antigen in Graves' ophthalmopathy (P less than 0.01). Positive leucocyte adherence inhibition was observed in 9 out of 21 cases of Graves' disease without ophthalmopathy, but its prognostic relevance has to be confirmed in the development of ophthalmopathy.     

Immunization with FimA protects against Streptococcus parasanguis endocarditis in rats.

FimA, a surface-associated protein of Streptococcus parasanguis, is associated with initial colonization of damaged heart tissue in an endocarditis model (D. Burnette-Curley, V. Wells, H. Viscount, C. Munro, J. Fenno, P. Fives-Taylor, and F. Macrina, Infect. Immun. 63:4669-4674, 1995). We have evaluated the efficacy of recombinant FimA as a vaccine in the rat model of endocarditis and investigated in vitro the mechanism for the protective role of immunization. FimA-immunized and nonimmunized control animals were catheterized to induce heart valve damage and infected intravenously with 10(7) CFU of wild-type S. parasanguis FW213 bacteria. The presence of bacteria associated with platelet-fibrin vegetations 24 h postchallenge was evaluated. Immunized rats were significantly less susceptible to endocarditis (2 cases among 34 animals) than the control group (21 cases among 33 animals) (P < 0.001). Incubation of S. parasanguis FW213 with rabbit anti-FimA immune serum decreased the mean percent adherence (0.34% of added cells) to platelet-fibrin matrix in vitro compared with that of preimmune normal serum (5.04% of added cells; P < 0.001). Adsorption of immune serum with FimA-positive S. parasanguis FW213 yielded antiserum that failed to block adherence to the platelet-fibrin matrix. We assessed the vaccine potential of FimA as a common immunogen able to provide cross-protection in streptococcal endocarditis by determining the occurrence and expression of fimA in the viridans group streptococci and enterococci. We detected the presence of fimA homologs by Southern hybridization and PCR amplification analyses and determined by immunoblotting the expression of FimA-like proteins among a variety of streptococci and enterococci that frequently cause endocarditis. Eighty-one percent (26 of 32) of streptococcal and enterococcal strains isolated from bacteremic patients expressed proteins that comigrated with FimA and were reactive with polyclonal anti-FimA serum. Streptococcal DNA from strains that were positive by Western blot (immunoblot) analysis hybridized to the full-length fimA probe. Our studies suggest that FimA immunization results in antibody-mediated inhibition of bacterial adherence, a critical early event in the pathogenesis of endocarditis. Our data demonstrate that a majority of streptococcal strains associated with endocarditis have genes that encode FimA-like proteins. Taken together, these results suggest that FimA is a promising candidate for an endocarditis vaccine.     

Enzyme-linked immunosorbent assay for detection of Staphylococcus epidermidis antibody in experimental S. epidermidis endocarditis.

The immune response against Staphylococcus epidermidis, as determined by an enzyme-linked immunosorbent assay, was evaluated in experimental S. epidermidis infections in rabbits. Antigens from 8 of 10 clinical S. epidermidis strains detected significant antibody production in five rabbits immunized with different strains of S. epidermidis and in five of six rabbits with experimental endocarditis caused by four different strains. The antigens from two strains detected antibody production in all rabbits, and strain ATCC 14990 discriminated best between positive and negative samples. Consecutive blood samples from rabbits with endocarditis and control rabbits with bacteremia, which were successfully prevented from developing endocarditis by using prophylactic antibiotics, were examined by using an enzyme-linked immunosorbent assay and an ultrasonic extract of strain ATCC 14990 as the antigen. This assay discriminated between rabbits with endocarditis and rabbits with uncomplicated bacteremia. Antibody production was detected as early as 3 days after the onset of infection in rabbits with endocarditis.     

Adherence of human peripheral blood lymphycytes to measles-infected cells. Enhancement by prostaglandin E1.

We studied the effect of prostaglandins in vitro on an immune reaction mediated by T cells; adherence of lymphocytes to measles-virus-infected human epithelial cells. Normal human lymphocytes adhered to a mean +/- S.D. 20.1 +/- 5.2 per cent of these HEp-2 cells. The percentage positive cells increased to 50.3 +/- 5.7 when lymphocytes were incubated with 10(-6) M prostaglandin E1 (P less than 0.01 vs. untreated lymphocytes); 10(-8) M and 10(-10) M were as effective. Prostaglandin F2alpha had no effect on lymphocyte adherence. Prostaglandin E1 increased lymphocyte cyclic AMP five to 10 times whereas prostagladin F2alpha did not affect cellular levels of this nucleotide. Dibutyryl cyclic AMP (10(-8) M) increased lymphocyte adherence: positive human epithelial cells increased from 16.0 +/- 2.4 to 38.7 +/- 1.1 per cent (P less than 0.01). Prostagladin E1 also increased adherence of lymphocytes from patients with systemic lupus erythematosus from 21.8 +/- 5.8 to 52.5 +/- 9.2 per cent (P less than 0.01). These results indicate that prostagladin E1 and cyclic AMP may serve to stimulate T-cell function and cell-mediated immunity.     

Role of Staphylococcus aureus coagulase and clumping factor in pathogenesis of experimental endocarditis.

The pathogenic role of staphylococcal coagulase and clumping factor was investigated in the rat model of endocarditis. The coagulase-producing and clumping factor-producing parent strain Staphylococcus aureus Newman and a series of mutants defective in either coagulase, clumping factor, or both were tested for their ability (i) to attach in vitro to either rat fibrinogen or platelet-fibrin clots and (ii) to produce endocarditis in rats with catheter-induced aortic vegetations. In vitro, the clumping factor-defective mutants were up to 100 times less able than the wild type strain to attach to fibrinogen and also significantly less adherent than the parents to platelet-fibrin clots. Coagulase-defective mutants, in contrast, were not altered in their in vitro adherence phenotype. The rate of in vivo infection was inoculum dependent. Clumping factor-defective mutants produced ca. 50% less endocarditis than the parent organisms when injected at inoculum sizes infecting, respectively, 40 and 80% (ID40 and ID80, respectively) of rats with the wild-type strain. This was a trend at the ID40 but was statistically significant at the ID80 (P < 0.05). Coagulase-defective bacteria were not affected in their infectivity. Complementation of a clumping factor-defective mutant with a copy of the wild-type clumping factor gene restored both its in vitro adherence and its in vivo infectivity. These results show that clumping factor plays a specific role in the pathogenesis of S. aureus endocarditis. Nevertheless, the rate of endocarditis with clumping factor-defective mutants increased with larger inocula, indicating the contribution of additional pathogenic determinants in the infective process.     

Reassessing the role of Staphylococcus aureus clumping factor and fibronectin-binding protein by expression in Lactococcus lactis.

Since Staphylococcus aureus expresses multiple pathogenic factors, studying their individual roles in single-gene-knockout mutants is difficult. To circumvent this problem, S. aureus clumping factor A (clfA) and fibronectin-binding protein A (fnbA) genes were constitutively expressed in poorly pathogenic Lactococcus lactis using the recently described pOri23 vector. The recombinant organisms were tested in vitro for their adherence to immobilized fibrinogen and fibronectin and in vivo for their ability to infect rats with catheter-induced aortic vegetations. In vitro, both clfA and fnbA increased the adherence of lactococci to their specific ligands to a similar extent as the S. aureus gene donor. In vivo, the minimum inoculum size producing endocarditis in > or =80% of the rats (80% infective dose [ID80]) with the parent lactococcus was > or =10(7) CFU. In contrast, clfA-expressing and fnbA-expressing lactococci required only 10(5) CFU to infect the majority of the animals (P < 0.00005). This was comparable to the infectivities of classical endocarditis pathogens such as S. aureus and streptococci (ID80 = 10(4) to 10(5) CFU) in this model. The results confirmed the role of clfA in endovascular infection, but with a much higher degree of confidence than with single-gene-inactivated staphylococci. Moreover, they identified fnbA as a critical virulence factor of equivalent importance. This was in contrast to previous studies that produced controversial results regarding this very determinant. Taken together, the present observations suggest that if antiadhesin therapy were to be developed, at least both of the clfA and fnbA products should be blocked for the therapy to be effective.     

FimA, a major virulence factor associated with Streptococcus parasanguis endocarditis.

Adherence of microorganisms to damaged heart tissue is a crucial event in the pathogenesis of infective endocarditis. In the present study, we investigated the role of the FimA protein as a potential virulence factor associated with Streptococcus parasanguis endocarditis. FimA is a 36-kDa surface protein that is a recognized adhesin in the oral cavity where it mediates adherence to the salivary pellicle. An insertion mutant and a deletion mutant of S. parasanguis were employed in the rat model of endocarditis to determine the relevance of FimA in endocarditis pathogenesis. Catheterized rats were infected with either the fimA deletion mutant VT929, the fimA insertion mutant VT930, or the isogenic, wild-type S. parasanguis FW213. Rats inoculated with FW213 developed endocarditis more frequently (50.9%) than animals inoculated with either the deletion mutant (2.7%) or the insertion mutant (7.6%) (P < 0.001). A series of in vitro assays were performed to explore the mechanism(s) by which FimA enhanced the infectivity of S. parasanguis. FimA did not inhibit the uptake or the subsequent killing of S. parasanguis by phagocytic granulocytes. Similarly, FimA did not play a role in the adherence to or the aggregation of platelets. Significant differences were noted between FW213 and VT929 (P < 0.05) and FW213 and VT930 (P < 0.001) in their abilities to bind to fibrin monolayers. The mean percent adherence of FW213 to fibrin monolayers (2.1%) was greater than those of VT929 (0.5%) and VT930 (0.12%). Taken together, these results indicate that FimA is a major virulence determinant associated with S. parasanguis endocarditis and further suggest that its role is associated with initial colonization of damaged heart tissue.     

Immune adherence and clearance of hepatitis B surface Ag/Ab complexes is abnormal in patients with systemic lupus erythematosus (SLE).

Complement levels and complement receptor 1 (CR1) on erythrocytes (E) are reduced in systemic lupus erythematosus (SLE). To see whether these abnormalities are responsible for defective transport and elimination of immune complexes (IC) from the circulation, patients with active SLE (14) and normal volunteers (14) were injected with preformed IC (hepatitis B surface Ag/Ab). Two minutes after injection only 25.9 +/- 19.1% (mean +/- 1 s.d.) of the circulating IC were bound to E in the SLE patients as compared to 63 +/- 3.7% in the normal subjects (P = 0.0001). For SLE patients, the reduced immune adherence was best explained by a combination of complement depletion and low CR1 binding capacity (tau = 0.80, P = 0.0001). The disappearance of IC as estimated from the area under the elimination curve was faster in SLE than in controls (P = 0.02), and correlated with CR1 (tau = 0.54, P = 0.0001) and immune adherence observed in vivo (tau = 0.33, P = 0.013). Finally, immune adherence was absent and IC disappeared very rapidly in a patient with C2 deficiency and an SLE-like disease. These observations suggest that in SLE the defective immune adherence reaction might be responsible for the accelerated disappearance of IC from the circulation.     

Role of immunoblotting in the diagnosis of culture negative and enterococcal endocarditis.

Serum samples from patients with endocarditis and septicaemia due to Enterococcus faecalis, Enterococcus faecium, Streptococcus bovis, and Streptococcus sanguis were immunoblotted against antigenic extracts from all four species. In E faecalis endocarditis there was a strong IgM response to E faecalis antigenic bands of 112, 88-90, and 45-47 Kd and a strong IgG response to 88-90 and 45-47 Kd bands. In E faecium endocarditis there was a pronounced IgG response to an E faecium band of 82-90 Kd. For S bovis endocarditis, there was a strong IgG response to several components of S bovis including bands of 66, 58, 52 and 4 Kd. For S sanguis, there was a strong IgG response to bands of 80-82, 76, 60 and 45 Kd. These patterns of antibody production were absent in patients with uncomplicated septicaemia and in controls. The delineation of these patterns enabled confirmation of the final diagnosis in seven patients initially suspected of having culture negative endocarditis.     

Multiple receptors on human monocytes are involved in adhesion to cultured human endothelial cells

Monocytes exhibit significant basal (unstimulated) adherence to human umbilical vein endothelium (HUVE), which is only partially inhibited by an anti-CD18 monoclonal antibody (mAb) (60.3). We examined factors modulating the residual, CD18-independent monocyte binding to HUVE by pretreating monocytes with mAb 60.3 to eliminate CD18-dependent binding. Basal adherence was reduced from 32% +/- 2% to 14% +/- 2% with mAb 60.3 (means +/- SE of eight experiments; P less than 0.01). mAb 60.3-treated monocytes were incubated with tumor necrosis factor-gamma (TNF-alpha), interleukin-1 (IL-1), lipopolysaccharide (LPS), N-formylmethionyl-leucyl-phenylalamine (FMLP), or phorbol myristate acetate (PMA). Only PMA affected CD18-independent binding. Pretreatment with PMA alone reduced adherence to 21% +/- 2% (mean +/- SE of eight experiments; P less than 0.01). In conjunction with mAb 60.3, PMA virtually eliminated monocyte adherence to HUVE (7% +/- 1%, mean +/- SE of eight experiments; P less than 0.01). We also examined CD18-independent monocyte binding to endothelial-leukocyte adhesion molecules (E-LAMs) induced by pretreatment of HUVE with LPS. Monoclonal antibody 60.3-treated monocytes increased adherence from 14% +/- 2% with unstimulated HUVE to 37% +/- 2% with LPS-stimulated HUVE (mean +/- SE of four experiments; P less than 0.01). Monocytes pretreated with both mAb 60.3 and PMA increased adherence from 5% +/- 1% with the unstimulated HUVE to 18% +/- 1% with the LPS-stimulated HUVE (mean +/- SE of four experiments; P less than 0.01). This result implies the presence of a CD18-independent and PMA-insensitive receptor on human monocytes for an E-LAM induced by LPS. In summary, we have identified two CD18-independent mechanisms of monocyte adherence to HUVE; a PMA-sensitive mechanism mediating basal adherence and a PMA-insensitive mechanism involved in binding to E-LAMs.     

Role of the Vegetation in Experimental Streptococcus viridans Endocarditis

This study examines the role of the vegetation in catheter-induced experimental endocarditis in predisposing to bacterial colonization of cardiac valves and in influencing the course of the disease and response to penicillin therapy. Platelet-fibrin vegetations developed at areas of valvular trauma and were colonized when Streptococcus viridans were injected intravenously. Pretreatment with warfarin prevented vegetation formation, but animals still developed endocarditis at the same rate after injection of 10(6)S. viridans. The course of the disease in anticoagulated animals was more explosive, as determined by a more rapid rise in fever and level of bacteremia. Mean survival was shorter in anticoagulated rabbits (7 versus 12.7 days). Large vegetations containing 10(9)S. viridans/g were found in control animals, whereas anticoagulated rabbits developed only microscopic deposits. Large vegetations required a longer duration of penicillin therapy to sterilize than the infected valves of the anticoagulated group (7 versus 3 days). Therefore, a preformed platelet-fibrin deposit is not a prerequisite for bacterial colonization of cardiac valves. After infection, the vegetation is an important factor in determining the subacute course of disease and resistance to penicillin therapy.