Effects of interleukin-1, lipopolysaccharide, and streptococci on procoagulant activity of cultured human cardiac valve endothelial and stromal cells.

Fibrin is the primary constituent of the vegetation in infective endocarditis, and tissue factor expression is a major mechanism of coagulation activation on infected valves. To determine which cells may participate in coagulation activation in this setting, expression of procoagulant activity (PCA; shown to be tissue factor) was studied in cultured endothelial and stromal cells derived from human cardiac valves. Endothelial cells had negligible PCA (99 +/- 50 mU/10(5) cells, mean +/- 1 standard deviation) unless stimulated by lipopolysaccharide or interleukin-1, which increased PCA to 5,592 +/- 1,482 and 5,901 +/- 1,497 mU/10(5) cells, respectively, in 6 h. Incubation of cells with viable enterococci or viridans streptococci or with an enterococcal cell wall preparation did not induce PCA. Cultured valve stromal cells constitutively expressed high levels of PCA (14,276 +/- 8,738 mU/10(5) cells) which was not changed with exposure to interleukin-1. PCAs of stromal or stimulated endothelial cells from valves of both right and left sides of the heart were comparable. The results suggest that endothelial cells may contribute to fibrin deposition during infection if stimulated, but PCA is not directly induced by bacteria. Stromal cells could contribute PCA if exposed to blood in the course of valve injury.     

Invasion and killing of human endothelial cells by viridans group streptococci

Colonization of the cardiovascular endothelium by viridans group streptococci can result in infective endocarditis and possibly atherosclerosis; however, the mechanisms of pathogenesis are poorly understood. We investigated the ability of selected oral streptococci to infect monolayers of human umbilical vein endothelial cells (HUVEC) in 50% human plasma and to produce cytotoxicity. Planktonic Streptococcus gordonii CH1 killed HUVEC over a 5-h period by peroxidogenesis (alpha-hemolysin) and by acidogenesis but not by production of protein exotoxins. HUVEC were protected fully by addition of supplemental buffers and bovine liver catalase to the culture medium. Streptococci were also found to invade HUVEC by an endocytic mechanism that was dependent on polymerization of actin microfilaments and on a functional cytoskeleton, as indicated by inhibition with cytochalasin D and nocodazole. Electron microscopy revealed streptococci attached to HUVEC surfaces via numerous fibrillar structures and bacteria in membrane-encased cytoplasmic vacuoles. Following invasion by S. gordonii CH1, HUVEC monolayers showed 63% cell lysis over 4 h, releasing 64% of the total intracellular bacteria into the culture medium; however, the bacteria did not multiply during this time. The ability to invade HUVEC was exhibited by selected strains of S. gordonii, S. sanguis, S. mutans, S. mitis, and S. oralis but only weakly by S. salivarius. Comparison of isogenic pairs of S. gordonii revealed a requirement for several surface proteins for maximum host cell invasion: glucosyltransferase, the sialic acid-binding protein Hsa, and the hydrophobicity/coaggregation proteins CshA and CshB. Deletion of genes for the antigen I/II adhesins, SspA and SspB, did not affect invasion. We hypothesize that peroxidogenesis and invasion of the cardiovascular endothelium by viridans group streptococci are integral events in the pathogenesis of infective endocarditis and atherosclerosis.     

Newly recognized distinctive M protein associated with certain M type 1 group A streptococci.

Immunological and epidemiological characteristics of M1T1 and M1T8 (T8, 25, Imp.19) group A streptococci isolated in Egypt have been described. Both strains possess a common M1 antigen. The M1T8 strains possess a second antigen which has the immunological and biological characteristics of M protein. The M1T1 strains do not possess this second M antigen. Absorbed rabbit antisera which are specific for this second antigen do not react with extracts of any of the known M protein serotypes. This second antigen is therefore a newly recognized M protein. The M1T1 and M1T8 strains had mutually exclusive geographical and temporal distributions. This finding may have been a consequence of the immunity which occurred after the initial occurrence of the M1T8 strain.     

Both plasmacytoid dendritic cells and monocytes stimulate natural killer cells early during human herpes simplex virus type 1 infections

Herpes simplex virus type 1 (HSV‐1), a member of the herpes virus family, is characterized by a short replication cycle, high cytopathogenicity and distinct neurotropism. Primary infection and reactivation may cause severe diseases in immunocompetent and immunosuppressed individuals. This study investigated the role of human plasmacytoid dendritic cells (pDC) in the activation of natural killer (NK) cells for the control of herpesviral infections. Within peripheral blood mononuclear cells, UV‐inactivated HSV‐1 and CpG‐A induced CD69 up‐regulation on NK cells, whereas infectious HSV‐1 was particularly active in inducing NK cell effector functions interferon‐γ (IFN‐γ) secretion and degranulation. The pDC‐derived IFN‐α significantly contributed to NK cell activation, as evident from neutralization and cell depletion experiments. In addition, monocyte‐derived tumour necrosis factor‐α (TNF‐α) induced after exposure to infectious HSV‐1 was found to stimulate IFN‐γ secretion. A minority of monocytes was shown to be non‐productively infected in experiments using fluorescently labelled viruses and quantitative PCR analyses. HSV‐1‐exposed monocytes up‐regulated classical HLA‐ABC and non‐classical HLA‐E molecules at the cell surface in an IFN‐α‐dependent manner, whereas stress molecules MICA/B were not induced. Notably, depletion of monocytes reduced NK cell effector functions induced by infectious HSV‐1 (P < 0·05). Altogether, our data suggest a model in which HSV‐1‐stimulated pDC and monocytes activate NK cells via secretion of IFN‐α and TNF‐α. In addition, infection of monocytes induces NK cell effector functions via TNF‐α‐dependent and TNF‐α‐independent mechanisms. Hence, pDC and monocytes, which are among the first cells infiltrating herpetic lesions, appear to have important bystander functions for NK cells to control these viral infections.     

Induction of procoagulant activity on human endothelial cells by Streptococcus pneumoniae.

The inflammatory response in infection caused by gram-negative organisms involves induction of procoagulant activity (PCA) on human endothelial cells. Although infections caused by gram-positive organisms are also associated with fibrin formation and thrombosis, the bacterial determinants inducing PCA are unknown. This study shows that intact pneumococci and the pneumococcal cell wall efficiently induce PCA on human endothelial cells. Upon exposure of endothelial cells to pneumococci, PCA was first detectable at 30 min, peaked at 2 h, and disappeared by 6 h. The specific activities of encapsulated and unencapsulated strains for induction of PCA were equivalent. Purified pneumococcal cell walls were as potent as endotoxin in induction of PCA. The ability to induce a procoagulant state on endothelial cells is a new biological activity of gram-positive cell walls which promotes the participation of the coagulation cascade in the inflammatory response to disease caused by gram-positive organisms.     

A human IgG 3 is opsonicin vitro against type III group B streptococci

Preparations of IgG 3 isolated by absorption of IgG 1, IgG 2, and IgG 4 from a human iv immunoglobulin with protein A-Sepharose were evaluated for their opsonic activities against type III group B streptococcal (GBS) strains. The resulting preparations were free of IgG 1 and IgG 2 and contained only trace amounts of IgG 4 (<2% of total IgG). These IgG 3 preparations exhibited excellent opsonic activities against type III GBS strains, similar to those of the unfractionated iv immunoglobulin (based on total IgG concentrations in the opsonic assays). In contrast, preparations of IgG 1, 2, and 4 eluted from protein A-Sepharose with 2M acetic acid and 7M urea were significantly less effective in enhancing phagocytosis and killing of type III GBS than IgG 3 preparations or iv immunoglobulin. The reasons for excellent opsonic activity of IgG 3 preparations as well as for decreased opsonic activity of IgG 1, 2, and 4 preparations are not clear. Perhaps alteration of IgG by lower pH and high concentrations of urea may have impaired the functional activity of IgG 1, 2, and 4 preparations. The significant finding of this study is the first demonstration of the excellent opsonic activity of IgG 3, emphasizing the importance of having intact IgG 3 in commercial immunoglobulin preparations used in prophylaxis or treatment of GBS infections.     

Biology and pathogenesis of thrombosis and procoagulant activity in invasive infections caused by group A streptococci and Clostridium perfringens

Group A streptococcal necrotizing fasciitis/myonecrosis and Clostridium perfringens gas gangrene are two of the most fulminant gram-positive infections in humans. Tissue destruction associated with these infections progresses rapidly to involve an entire extremity. Multiple-organ failure is common, and morbidity and mortality remain high. Systemic activation of coagulation and dysregulation of the anticoagulation pathways contribute to the pathogenesis of many diverse disease entities of infectious etiology, and it has been our hypothesis that microvascular thrombosis contributes to reduced tissue perfusion, hypoxia, and subsequent regional tissue necrosis and organ failure in these invasive gram-positive infections. This article reviews the coagulation, anticoagulation, and fibrinolytic systems from cellular players to cytokines to novel antithrombotic therapies and discusses the mechanisms contributing to occlusive microvascular thrombosis and tissue destruction in invasive group A streptococcal and C. perfringens infections. A thorough understanding of these mechanisms may suggest novel therapeutic targets for patients with these devastating infections.     

Influenza type A virus-mediated adherence of type 1a group B streptococci to mouse tracheal tissue in vivo.

Influenza type A virus-mediated adherence of pathogenic bacteria to the mucosal surface of the respiratory tract may be one of several mechanisms whereby influenza predisposes to bacterial pneumonia. In the present study, we quantified the adherence of intranasally administered type 1a group B streptococci to the tracheal tissue of influenza type A/PR8/34 (HONI) virus-infected and mock-infected mice. Influenza type A/PR8/34 virus infection effected a 120-fold increase in the adherence of type 1a group B streptococci to tracheal tissue relative to that observed in mock-infected mice. Adherence of type 1a group B streptococci to the trachea of influenza type A/PR8/34 virus-infected mice was reduced by more than 90% by prior intranasal instillation of chicken antiserum to influenza type A/PR8/34 virus, whereas virtually no reduction in adherence was noted with normal chicken serum or rabbit antiserum to herpes simplex virus type 2. These findings suggest that adherence of type 1a group B streptococci to the tracheal tissue of influenza type A/PR8/34 virus-infected mice is effected by a viral component(s).     

Genetic heterogeneity of M type 3 group A streptococci causing severe infections in Tayside, Scotland.

To explain the worldwide increase in the frequency of severe infections by group A streptococci, molecular techniques are increasingly being employed to evaluate the genetic relationships of strains. We used restriction endonuclease analysis, pulsed-field gel electrophoresis (PFGE), the PCR, ribotyping, and DNA sequence analysis in a study of 13 group A streptococci isolated from a cluster of cases of serious infections over a 3-month period in Tayside, Scotland. Eight of the strains were M type 3; molecular characterization identified two subclones. The first, displaying PFGE profile 4, has been observed in Northern Scotland and has been circulating in New Zealand for over a decade. The second subclone has been documented only in the United Kingdom; it was first seen in 1993 in Scotland. Sequence analysis of emm-3 genes further differentiated the PFGE 4 subclone. DNA sequence analysis of virulence factors supports the suggestion that they may have recently been acquired by horizontal gene transfer.     

Effect of cyclosporine A on procoagulant activity in mononuclear blood cells and monocytes in vitro.

Monocytes and macrophages synthesize thromboplastin when induced by a number of compounds. Our experiments show that cyclosporine A (CyA) enhances the synthesis of procoagulant activity in monocytes and in whole mononuclear cells (MNC) about two-fold when phytohaemagglutinin (PHA) is the stimulant. The enhancement is inhibited by actinomycin D and cycloheximide. CyA alone had no effect, but in the presence of PHA or phorbol ester CyA induces or enhances the synthesis of a vitamin K-dependent procoagulant in the MNC cultures. This procoagulant acts synergistically with thromboplastin synthesized in the same cultures.     

Unusual occurrence of M type 77, antibiotic-resistant group A streptococci in southern Sweden

For many years group A streptococci of T type 28 (T28) have been common in southern Sweden; however, since 1995 resistance to both macrolide-lincosamide-streptogramin B (MLS) antibiotics and tetracycline was observed among T28 isolates, which prompted the present studies on clonal relatedness of antibiotic-resistant T28 strains. By extended T typing, 95 of 100 examined tetracycline-resistant strains showed the combination T9-T13-T28; of these, 94 belonged to M type 77 (M77) and one belonged to M73. Three strains were T28-M28 and two were T28-M nontypeable. The serological M77 was confirmed by PCR capture enzyme-linked immunosorbent assay, emm amplicon restriction profiling, and emm sequence typing. Fifty strains were examined for superantigen genes: speA was detected in three blood isolates only, whereas all isolates harbored speB, and only two of the strains were negative for speC. Eighty-nine of the 100 strains were also macrolide resistant, of which 59 were inducibly MLS resistant (IR) and 21 were constitutively MLS resistant (CR), 6 were noninducibly resistant (NI), and 3 had novel subphenotypes recently reported by our group. Resistance genes were determined by PCR and hybridization methods. Eighty-four of the 100 strains harbored tetM. ermB was detected in all CR and IR strains, and mefA was found in all NI strains; both ermB and mefA were identified in two strains with novel subphenotypes. Pulsed-field gel electrophoresis showed that these antibiotic-resistant M77 strains belonged to at least five different clones.     

The procoagulant activity of human granulocytes,lymphocytes and monocytes stimulated by endotoxin

Summary Leukocytes from donor blood were separated by Ficoll/Urovison density centrifugation into granulocytes, lymphocytes and monocytes. The cell fractions were suspended in a culture medium to which endotoxin of Salmonella enteritidis was added at a final concentration of 10 µg/ml. Endotoxin-stimulated monocytes developed a very high tissue factor (thromboplastin) activity while in granulocytes an only negligible amount of tissue factor activity was detectable. The tissue factor activity measured in the preparation of the lymphocytes can be explained by contamination with monocytes. Electron microscopic studies showed the lysosomes of all monocytes to be enlarged and activated. Only a fraction of the granulocytes appeared degranulated with prominent vacuoles containing inclusion bodies. Possibly the high tissue factor activity of the monocytes triggers the development of the disseminated intravascular coagulation in the Shwartzman phenomenon.These investigations were supported in part by DFG Hi 224/1     

Biological and immunochemical identity of M protein on group G streptococci with M protein on group A streptococci.

Previous evidence for the presence of an M or M-like protein on group G streptococci has been based on the ability of these strains to survive in human blood. In addition, cross-reactions between group A and group G streptococci have been demonstrated, but they have relied either on whole bacterial cell vaccine-induced polyclonal sera or crude protein extracts of these cells. In this study two monoclonal antibodies prepared against the purified, native group A streptococcal M6 protein demonstrated a high degree of cross-reactivity with group G streptococcal clinical isolates (9 and 19 of 22 strains examined, respectively). Ten of these strains exhibited resistance to phagocytosis when rotated in human blood. In addition, immunoblot analysis of crude mutanolysin extracts of group G streptococci with one of the M6 monoclonal antibodies illustrated a remarkable similarity in the protein pattern of these extracts as compared with those of group A streptococcal M protein. The immunoblots further demonstrated a variation in the relative molecular weights of the extracted proteins from strain to strain over a range of 57,000 to 77,000. In addition, a purified, pepsin-derived fragment (Mr, 43,000) from a group G strain was capable of eliciting rabbit antibodies that were opsonic for group G cells in a bactericidal assay. These functional and immunochemical data, in concert with DNA hybridization between group G streptococcal DNA and a group A M6 gene probe (J. R. Scott, W. M. Pulliam, S. K. Hollingshead, and V. A. Fischetti, Proc. Natl. Acad. Sci. USA 82:1822-1826, 1985), provide strong evidence for the presence of an M protein on these organisms and indicate its probable role as a virulence molecule on the surface of group G streptococci.     

Nonimmune binding of human immunoglobulin A to type II group B streptococci

The binding of 125I-labeled human myeloma immunoglobulin A (IgA) to four type II strains and one nontypable strain of group B streptococci was measured after streptococcal chains were broken by brief sonication. Some IgA binding was observed with all strains, but specific binding (binding that was inhibited by excess unlabeled IgA, was dose dependent, and was saturable) occurred only with those strains possessing the trypsin-sensitive beta component of the c protein. Similar amounts of binding were observed with myeloma IgA and IgA1 purified from normal serum. Specific binding was more rapid at 25 degrees C than at 0 or 37 degrees C and reached a plateau in 6 to 8 h. Binding was drastically reduced (85 to 90%) when streptococci had been heated (90 degrees C for 1 h). Most radioactivity bound to group B streptococci could be displaced (greater than 60% in 3 days) by the addition of excess unlabeled IgA. The binding capacity of one strain (10(8) streptococci in 1 ml of buffer) was saturated by approximately 24 micrograms of IgA. When transformed for Scatchard analysis, these data indicated that there was a specific binding capacity of 16,000 molecules of monomeric serum IgA per single streptococcal cell. The dissociation constant for IgA binding was 19.3 nM. Since enzyme-linked immunosorbent assay studies showed that the myeloma IgA used for the studies described above was IgA1, our quantitative data apply only to the binding of this subclass to group B streptococci. However, an enzyme-linked immunosorbent-filtration assay showed that both IgA1 and IgA2 bound to a type II group B streptococcus bearing the c protein.     

Myocardial tissue damage in rabbits injected with group A streptococci, types M1 and M22. Role of bacterial immunoglobulin G-binding surface proteins

Acute rheumatic fever (ARF) and acute poststreptococcal glomerulonephritis (APSGN), two important sequelae of streptococcal throat or skin infections, according to current concepts may be elicited by autoimmune mechanisms due to molecular mimicry between group A streptococci (GAS) and human tissue. In the case of APSGN, however, our experimental data have indicated that GAS immunoglobulin-binding surface proteins (IgG BPs) might be of pathogenic significance by triggering anti-IgG production and immune complex formation leading to renal damage. Thus, rabbits injected with IgG-binding, as opposed to non-binding, GAS strains were found to develop renal deposition of IgG and complement factor C3 and inflammatory and degenerative glomerular changes resembling the picture seen in APSGN. In the present study, cardiac tissue material from rabbits injected with GAS was investigated. After 8 or more weeks of intravenous (i.v.) injections, minimal changes were seen in those animals receiving an IgG non-binding GAS strain, type T27, whereas those animals receiving either of two IgG-binding GAS strains, types M1 or M22, developed strong inflammatory and degenerative myocardial changes accompanied by deposition of IgG and C3. Furthermore, on injecting rabbits with defined mutants of a type M22 strain, the development of myocardial tissue damage proved to be dependent on the presence of streptococcal IgG-binding activity. Our results demonstrate that myocardial tissue changes may be induced in the rabbit by i.v. injection of whole heat-killed GAS of at least two M serotypes. Conceivably, induction of immune complexes by bacterial IgG BPs may lead to myocardial deposition of IgG, in turn triggering a series of events, involving the complement system and proinflammatory cytokines, with resulting tissue damage. Though many virulence factors may be involved in the development of ARF and APSGN, and a given GAS strain will never cause both, our results may suggest a new pathogenetic mechanism common to these two major non-suppurative complications.     

Opsonic effect of jacalin and human immunoglobulin A on type II group B streptococci.

This study examined the effect of immunoglobulin A (IgA) and the IgA-binding lectin jacalin on the phagocytosis of type II group B streptococci (GBS). Strains possessing the trypsin-sensitive and trypsin-resistant components of the c protein (II/c) and type II GBS lacking the c protein (II) were examined by radiolabeled bacterial uptake, bactericidal assays, and electron microscopy. Type II/c GBS resisted phagocytosis by monocytes (4.9% +/- 0.8% uptake, mean +/- SE, n = 25) compared with type II GBS (8.5% +/- 1.4% uptake, n = 14, P = 0.03). Phagocytic killing by polymorphonuclear leukocytes was also less for the type II/c strain 78-471 than for the type II strain 79-176 (68% +/- 5% versus 86% +/- 4% reduction in CFU at 45 min, P = 0.03). IgA binding did not explain the resistance of type II/c GBS to phagocytosis. The uptake of type II/c GBS was not significantly different after opsonization in cord sera lacking endogenous IgA (5.93% +/- 1.4%) than in the same cord sera after addition of exogenous IgA (5.48% +/- 1.4%, P = 0.69, n = 9). Attempts to remove serum IgA with the IgA-binding lectin jacalin resulted in the binding of IgA-jacalin complexes to II/c GBS. This combination of nonspecific IgA and jacalin increased uptake of II/c GBS from 4.9% +/- 0.8% to 11.8% +/- 1.9% (P = 0.002). Jacalin also combined with specific, immune, monoclonal IgA bound to the surface of Haemophilus influenzae and promoted the uptake of these bacteria. Jacalin and IgA mediated phagocytosis of II/c GBS via receptors that were not dependent on divalent cations and that were not modulated by plating monocytes on antigen-antibody complexes.     

Role of M protein in adherence of group A streptococci.

The role of the M protein in adherence of group A streptococci to human epithelial cells was directly tested by using an isogenic pair of M+ and M- strains. There was no difference between these strains in the number of streptococcal units that adhered to buccal or tonsillar epithelial cells, indicating the following: (i) that adhesins that are not dependent upon M protein expression are present on the surface of group A streptococci and (ii) that the M protein is not the primary streptococcal adherence ligand. However, the M+ strain adhered to tonsillar epithelial cells as aggregates. This aggregation was dependent on the presence of the M protein, since the isogenic M- strain did not clump. The coaggregation of streptococci suggests that the M protein plays an important role in promoting the formation of microcolonies after initial attachment. Binding to fibronectin, a potential epithelial cell receptor for group A streptococci, was also the same for the isogenic M+ and M- strains as well as for an isogenic strain with a regulatory mutation that decreases the expression of M protein. In summary, the M protein is not the primary streptococcal adhesin, nor is it required to orient the streptococcal adhesin and/or fibronectin receptor.     

Signal transduction and procoagulant state of human cord blood--progenitor-derived endothelial cells after interleukin-1alpha stimulation.

Isolation of endothelial progenitors from human umbilical cord blood generated great hope in vascular tissue engineering. However, before clinical use, progenitor derived endothelial cells (PDECs) have to be compared with mature endothelial cells (ECs). The aim of this study was to explore the behavior of PDECs exposed to a proinflammatory cytokine (interleukin-1alpha; IL-1alpha) according to the mitogen-activated protein (MAP) kinase and nuclear factor (NF)-kappaB signal transduction pathways as well as procoagulant activity (PCA). CD34(+) mononuclear cells were isolated using magnetic beads, cultured, and compared with human saphenous vein ECs (HSVECs). PDECs express endothelial markers: CD31, VE-cadherin, von Willebrand factor, KDR, and incorporate acetylated low-density lipoprotein (Dil-Ac-LDL). IL-1alpha similarly activates c-Jun N-terminal protein kinase (JNK) and p38 pathways in HSVECs and PDECs, whereas extracellular signal-related kinase (ERK)1/2 phosphorylation is lower in PDECs than in HSVECs. Low ERK1/2 phosphorylation in PDECs was specific to IL-1alpha as vascular endothelial growth factor (VEGF) similarly stimulated ERK1/2 pathway. With respect to inhibitor of NF-kappa B (Ikappa B) degradation, NF-kappa B translocation and phosphorylation, the NF-kappa B pathway is comparable in HSVECs and PDECs after stimulation. PCA and tissue factor level induced by IL-1alpha are lower in PDECs than in HSVECs. Thus, our data show that PDECs display the characteristics of functional mature ECs under IL-1alpha stimulation. However, we observed significant differences between PDECs and HSVECs related to both ERK1/2 pathway activation and tissue factor production.