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C E Rubens

Publications and source records attributed to C E Rubens.

At least 37 records · Page 2Linked to original sources
PubMed1998

Characterization of group B streptococcal invasion of human chorion and amnion epithelial cells In vitro.

Group B streptococci (GBS) have been cultured from the chorioamnionic membrane of pregnant women, usually in association with chorioamnionitis and premature labor (K. A. Boggess, D. H. Watts, S. L. Hillier, M. A. Krohn, T. J. Benedetti, and D. A. Eschenbach, Obstet. Gynecol. 87:779-784, 1996). Colonization and infection of placental membranes can be a prelude to neonatal GBS infections even in the presence of intact membranes (R. L. Naeye and E. C. Peters, Pediatrics 61:171-177, 1978), suggesting that GBS cause chorioamnionitis or establish amniotic fluid infections by partial or complete penetration of the placental membranes. We have isolated and grown cultures of primary chorion and amnion cells from human cesarean-section placentas. This has provided a biologically relevant model for investigating GBS adherence to and invasion of the two epithelial barriers of the placental membrane. GBS adhered to chorion cell monolayers to a high degree. Pretreatment of GBS with trypsin reduced adherence up to 10-fold, which suggested that the bacterial ligand(s) was a protein. GBS invaded chorion cells at a high rate in vitro, and invasion was dependent on cellular actin polymerization. GBS could be seen within intracellular vacuoles of chorion cells by transmission electron microscopy. We also demonstrated that GBS were capable of transcytosing through intact chorion cell monolayers without disruption of intracellular junctions. GBS also adhered to amnion cells; in contrast, however, these bacteria failed to invade amnion cells under a variety of assay conditions. GBS interactions with the chorion epithelial cell layer shown here correlate well with epidemiological and pathological studies of GBS chorioamnionitis. Our data also suggest that the amnion cell layer may provide an effective barrier against infection of the amniotic fluid.

Amnion↗
PubMed1997

The role of group B streptococci beta-hemolysin expression in newborn lung injury.

There is a direct correlation between the level of GBS beta-hemolysin expression and the ability of GBS to injury lung epithelial cells. Electron microscopy suggest the hemolysin acts as a pore-forming cytolysin. beta-hemolysin-associated lung epithelial cell injury is inhibited by surfactant phospholipid, a substance in which high-risk premature infants are deficient. We have now shown that loss of GBS hemolysin activity is associated with decreased animal virulence following intrathoracic inoculation of the organism. Further, a knockout of a putative GBS beta-hemolysin gene from the literature suggests it is not the major GBS hemolysin determinant. Cloning and sequencing analysis of the Tn916 (or Tn916DE) insertions in three of our nonhemolytic GBS mutants show identical integration sites in a distinct chromosomal locus. Finally, a putative 11-kd hemolysin species is identified by comparative analysis of protein extracts from isogenic hemolysin mutants.

Animals↗
PubMed1997

A role for C5 and C5a-ase in the acute neutrophil response to group B streptococcal infections.

Congenic C5-deficient and C5-sufficient mice were infected with group B streptococci (GBS) to determine if the polymorphonuclear leukocyte (PMNL) chemoattractant C5a contributes to PMNL recruitment in GBS infection and if GBS C5a-ase reduces C5a-induced PMNL recruitment in vivo. PMNL accumulation was greater in the peritoneum and air spaces of C5-sufficient mice than in C5-deficient mice. Administration of human C5 to C5-deficient mice caused a significant increase in PMNL recruitment following infection with C5a-ase-negative GBS. GBS C5a-ase did not reduce PMNL accumulation in C5-sufficient mice but reduced PMNL recruitment in C5-deficient mice reconstituted with human C5. These data indicate that C5a is important for rapid PMNL recruitment to sites of GBS infection and that GBS C5a-ase inactivates human, but not murine, C5a in vivo. Reduction of the acute inflammatory response by C5a-ase likely contributes to GBS virulence in human neonates.

Adhesins, Bacterial↗
PubMed1997

New genetic techniques for group B streptococci: high-efficiency transformation, maintenance of temperature-sensitive pWV01 plasmids, and mutagenesis with Tn917.

Three techniques were developed to improve the genetic manipulation of group B streptococci (GBS). We first optimized a protocol for transformation of GBS by electroporation, which provided transformation efficiencies of 10(5) CFU/microgram. Variables that influenced the transformation efficiency were the glycine content of the competent cell growth media, the electric field strength during electroporation, the electroporation buffer composition, the host origin of the transforming plasmid, and the concentration of selective antibiotic at the final plating. Our transformation protocol provides an efficiency sufficient for cloning from ligation reactions directly into GBS, obviating an intermediate host such as Escherichia coli. Second, temperature-sensitive plasmids of the pWV01 lineage were shown to transform GBS, and their temperature-sensitive replication was confirmed. Lastly, the temperature-sensitive pWV01 plasmid pTV1OK, which contains Tn917, was used as a transposon delivery vector for the construction of genomic Tn917 mutant libraries. We have shown, for the first time, that Tn917 transposes to the GBS chromosome and at a frequency of 10(-3)/CFU. Furthermore, representative clones from a Tn917 library contained single transposon insertions that were randomly located throughout the chromosome. These techniques should provide useful methods for cloning, mutagenesis, and characterization of genes from GBS.

DNA Replication↗
PubMed1997

Invasion of brain microvascular endothelial cells by group B streptococci.

Group B streptococci (GBS) are the leading cause of meningitis in newborns. Although meningitis develops following bacteremia, the precise mechanism or mechanisms whereby GBS leave the bloodstream and gain access to the central nervous system (CNS) are not known. We hypothesized that GBS produce meningitis because of a unique capacity to invade human brain microvascular endothelial cells (BMEC), the single-cell layer which constitutes the blood-brain barrier. In order to test this hypothesis, we developed an in vitro model with BMEC isolated from a human, immortalized by simian virus 40 transformation, and propagated in tissue culture monolayers. GBS invasion of BMEC monolayers was demonstrated by electron microscopy. Intracellular GBS were found within membrane-bound vacuoles, suggesting the organism induced its own endocytic uptake. GBS invasion of BMEC was quantified with a gentamicin protection assay. Serotype III strains, which account for the majority of CNS isolates, invaded BMEC more efficiently than strains from other common GBS serotypes. GBS survived within BMEC for up to 20 h without significant intracellular replication. GBS invasion of BMEC required active bacterial DNA, RNA, and protein synthesis, as well as microfilament and microtubule elements of the eukaryotic cytoskeleton. The polysaccharide capsule of GBS attenuated the invasive ability of the organism. At high bacterial densities, GBS invasion of BMEC was accompanied by evidence of cellular injury; this cytotoxicity was correlated to beta-hemolysin production by the bacterium. Finally, GBS demonstrated transcytosis across intact, polar BMEC monolayers grown on Transwell membranes. GBS invasion of BMEC may be a primary step in the pathogenesis of meningitis, allowing bacteria access to the CNS by transcytosis or by injury and disruption of the endothelial blood-brain barrier.

Brain↗
PubMed1996

Characterization of cpsF and its product CMP-N-acetylneuraminic acid synthetase, a group B streptococcal enzyme that can function in K1 capsular polysaccharide biosynthesis in Escherichia coli.

Group B Streptococcus (GBS) is the foremost cause of neonatal sepsis and meningitis in the United States. A major virulence factor for GBS is its capsular polysaccharide, a high molecular weight polymer of branched oligosaccharide subunits. N-acetylneuraminic acid (Neu5Ac or sialic acid), at the end of the polysaccharide side chains, is critical to the virulence function of the capsular polysaccharide. Neu5Ac must be activated by CMP-Neu5Ac synthetase before it is incorporated into the polymer. We showed previously that a transposon mutant of a serotype III GBS strain which had no detectable capsular Neu5Ac was deficient in CMP-Neu5Ac-synthetase activity (Wessels et al., 1992). In this paper, we report the identification and characterization of cpsF, a gene interrupted by transposon insertion in the previously described Neu5Ac-deficient mutant. The predicted amino acid sequence of the cpsF gene product shares 57% similarity and 37% identity with CMP-Neu5Ac synthase encoded by the Escherichia coli K1 gene, neuA. The enzymatic function of the protein encoded by cpsF was established by cloning the gene E. coli under the control of the T7 polymerase/promoter. Lysates of E. coli in which the cpsF gene product was expressed, catalysed the condensation of CTP with Neu5Ac to form CMP-Neu5Ac. In addition, when CMP-Neu5Ac synthetase-deficient mutant of E. coli K1 was transformed with cpsF, K1 antigen expression was restored. We concluded that cpsF encodes CMP-Neu5Ac synthetase in type III GBS, and that the GBS enzyme can function in the capsule-synthesis of a heterologous bacterial species.

Antigens, Bacterial↗
PubMed1996

A virulent nonencapsulated Haemophilus influenzae.

Nontypeable Haemophilus influenzae strain INT1 was isolated from the blood of a young child with clinical signs of meningitis following acute otitis media. No immunologic or anatomic predisposition of this child for invasive bacterial infection with an unusual organism was documented. Sensitive ELISA proved the absence of intra- or extracellular capsular polysaccharide production by INT1 and Southern blot analysis confirmed the lack of an intact capsulation (cap) gene locus within the chromosome. Nevertheless, INT1 established bacteremia and meningitis in infant and weanling rat models of invasive H. influenzae infection. High-molecular-weight DNA isolated from INT1 was shown to confer an invasive phenotype on transformation of a nonencapsulated, avirulent laboratory strain of H. influenzae. Together these findings imply the presence of one or more as-yet-undiscovered, noncapsular virulence factors of H. influenzae that are capable of mediating invasive disease and resistance to immunologic clearance.

Amoxicillin↗
PubMed1996

Vitamin A therapy for children with respiratory syncytial virus infection: a multicenter trial in the United States.

BACKGROUND: High dose vitamin A therapy is effective in reducing morbidity and mortality associated with measles infection. Children with acute respiratory syncytial virus (RSV) infection have low serum vitamin A concentrations. METHODS: We performed a multicenter, randomized, placebo-controlled trial of high dose vitamin A therapy among 239 children 1 month to 6 years of age to determine whether high dose vitamin A therapy would reduce morbidity associated with RSV infection. RESULTS: There were no differences between the vitamin A and placebo recipients for most clinical outcomes; however, vitamin A recipients had-longer hospital stays than placebo recipients (5.0 days vs. 4.4 days, P = 0.01) after enrollment. This effect was significant for children who were older than 1 year (who also had received the highest doses of vitamin A), particularly among those at low risk for complications of RSV infection and those enrolled during the second study season. Serum retinol levels at enrollment were inversely correlated with severity of illness. CONCLUSIONS: We found no evidence of a beneficial effect of vitamin A for the treatment of RSV infection in children in the United States. There may be groups of children for which vitamin A has an adverse effect, resulting in longer hospital stays.

Child↗
PubMed1996

Group B streptococcal beta-hemolysin expression is associated with injury of lung epithelial cells.

Group B streptococci (GBS) are the leading cause of serious bacterial infection in newborns. Early-onset disease is heralded by pneumonia and lung injury, and the lung may serve as a portal of entry for GBS into the bloodstream. To examine a potential role for GBS beta-hemolysin in lung epithelial injury, five wild-type strains varying in beta-hemolysin expression were chosen, along with five nonhemolytic (NH) and five hyperhemolytic (HH) variants of these strains derived by chemical or transposon mutagenesis. Monolayers of A549 alveolar epithelial cells were exposed to log-phase GBS or stabilized hemolysin extracts of GBS cultures, and cellular injury was assessed by lactate dehydrogenase (LDH) release and trypan blue nuclear staining. Whereas NH strains produced no detectable injury beyond baseline (medium alone), hemolysin-producing strains induced LDH release from A549 cells in direct correlation to their ability to lyse sheep erythrocytes. HH strains were also associated with marked increases in trypan blue nuclear staining of A549 monolayers. The extent of LDH release produced by HH strains was significantly reduced in the presence of dipalmitoyl phosphatidylcholine, a known inhibitor of hemolysin and the major phospholipid component of human surfactant. Electron microscopic studies of A549 cell monolayers exposed to HH GBS mutants revealed global loss of microvillus architecture, disruption of cytoplasmic and nuclear membranes, and marked swelling of the cytoplasm and organelles. We conclude that GBS hemolysin expression correlates with lung epithelial cell injury and may be important in the initial pathogenesis of early-onset disease, particularly when pulmonary surfactant is deficient.

1,2-Dipalmitoylphosphatidylcholine↗
PubMed1995

Group A streptococcal necrotizing fasciitis complicating primary varicella: a series of fourteen patients.

We retrospectively reviewed the clinical course of group A Streptococcus necrotizing fasciitis complicating primary varicella in children admitted to Children's Hospital and Medical Center, Seattle, WA, during a 18-month period. The potential benefit of various therapeutic interventions was examined. Fourteen children ages 6 months to 10 years were treated for group A Streptococcus necrotizing fasciitis as a complication of primary varicella. Eight patients experienced a delay in initial diagnosis as a result of nonspecific, early clinical findings of necrotizing fasciitis. Each patient underwent surgical exploration with fasciotomies and debridement. Initial antibiotic therapy was broad spectrum and included clindamycin. Hyperbaric oxygen therapy for as many as 6 treatments was used as adjunctively therapy in 12 patients, with subjective benefit in 6 patients. All 14 patients were discharged home with good function and no long term sequelae. This potentially fatal bacterial infection of the deep fascial layers requires early recognition by primary care physicians and an intensive, multidisciplinary therapeutic approach, including thorough surgical debridement and appropriate antibiotic therapy.

Anti-Bacterial Agents↗
PubMed1995

Group B streptococci adhere to a variant of fibronectin attached to a solid phase.

Group B streptococci (GBS) are the leading cause of neonatal pneumonia and meningitis. Adherence of GBS to host tissues may play an important role in the pathogenesis of infection. The host molecules which mediate GBS adherence to host tissues are unknown. Many bacterial pathogens adhere to fibronectin, an important component of the extracellular matrix (ECM). Some pathogens adhere to both immobilized and soluble fibronectin, while others adhere to immobilized fibronectin, but not to soluble fibronectin. Previous data indicated that GBS do not adhere to soluble fibronectin. We studied the ability of GBS to adhere to immobilized fibronectin. Forty-five per cent of the input inoculum of COH1, a virulent GBS isolate, adhered to fibronectin immobilized on polystyrene. COH1 did not adhere to the other ECM proteins tested (laminin, type I collagen, vitronectin, and tenascin). Nine out of nine GBS strains from human sources tested adhered specifically to fibronectin at levels varying from 4-60%. We considered the possibility that GBS were adherent to a contaminant in the fibronectin preparation. Properties of fibronectin, including the presence of an immunologic epitope of fibronectin and binding to collagen, were verified to be properties of the molecule to which GBS adhere. COH1 adhered to fibronectin captured by a monoclonal antibody to fibronectin (FN-15), confirming that the molecule to which GBS adhere bears immunologic determinants of fibronectin. Adherence of COH1 to fibronectin was inhibited by collagen, confirming that the molecule to which GBS adhere binds to collagen. These data strongly suggest that GBS adhere to fibronectin, and not to a contaminant.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Group B streptococci (GBS) injure lung endothelium in vitro: GBS invasion and GBS-induced eicosanoid production is greater with microvascular than with pulmonary artery cells.

Neonatal group B streptococcal (GBS) sepsis and pneumonia cause lung endothelial cell injury. GBS invasion of the lung endothelium may be a mechanism for injury and the release of vasoactive eicosanoids. Pulmonary artery endothelial cells (PAEC) and lung microvascular endothelial cells (LMvEC) were isolated from neonatal piglets and were characterized as endothelial on the basis of morphology, uptake of acyl low-density lipoprotein, factor VIII staining, and formation of tube-like structures on Matrigel. PAEC and LMvEC monolayers were infected with COH-1 (parent GBS strain), isogenic mutants of COH-1 devoid of capsular sialic acid or all capsular polysaccharide, or a noninvasive Escherichia coli strain, DH5 alpha. Intracellular GBS were assayed by plate counting of colony-forming units resistant to incubation with extracellular antibiotics. All GBS strains invaded LMvEC significantly more than PAEC, showing that the site of lung endothelial cell origin influences invasion. DH5 alpha was not invasive in either cell type. Both isogenic mutants invaded PAEC and LMvEC more than COH-1 did, showing that GBS capsular polysaccharide attenuates invasion. Live GBS caused both LMvEC and PAEC injury as assessed by lactate dehydrogenase release; heat-killed GBS and DH5 alpha caused no significant injury. Supernatants from PAEC and LMvEC were assayed by radioimmunoassay for prostaglandin E2 (PGE2), the stable metabolite of prostacyclin (6-keto-PGF1 alpha), and the thromboxane metabolite thromoxane B2. At 4 h, live COH-1 caused no significant increases in eicosanoids from both PAEC and LMvEC. At 16 h, live COH-1, but not heat-killed COH-1, caused a significant increase in 6-keto-PGF1 alpha greater than PGE2 from LMvEC, but not PAEC. We conclude that live GBS injure and invade the lung microvascular endothelium and induce release of prostacyclin and PGE2. We postulate that GBS invasion and injury of the lung microvasculature contribute to the pathogenesis of GBS disease.

Animals↗
PubMed1994

Adherence of group B streptococci to cultured epithelial cells: roles of environmental factors and bacterial surface components.

Group B streptococci (GBS) are the major cause of neonatal pneumonia, sepsis, and meningitis. Steps considered to be important in the pathogenesis of this infection include colonization of the rectum and vagina of the mother, aspiration of GBS into the fetal lung during or just prior to delivery, and invasion of GBS into pulmonary epithelial cells. We have previously demonstrated that GBS can invade pulmonary epithelial cells both in vivo and in vitro. Adherence of GBS to epithelial cells may play an important role in colonization of the rectum and vagina and constitute a first step in invasion of pulmonary epithelial cells. Because GBS can both adhere to and invade epithelial cells, we have developed two assays for GBS adherence which measure cell surface and not intracellular bacteria. Using these assays, we were able to demonstrate specific adherence of GBS to pulmonary epithelial cells. Adherence levels were similar at 4 and 37 degrees C and for log- and stationary-phase bacteria. Physiologic conditions vary considerably between the rectum, vagina, and lung, and a range of conditions was therefore tested. Adherence was enhanced in hypotonic solutions, while magnesium and calcium had no effect on adherence at physiologic concentrations. In comparison with adherence at neutral pH, adherence was increased 6- to 20-fold at pH 4, which is the normal vaginal pH. Neither capsular polysaccharide nor lipoteichoic acid was important for adherence in these assays. Treatment of GBS with trypsin decreased their adherence by more than 75%, indicating that surface proteins play an important role.

Bacterial Adhesion↗
PubMed1993

Production of tumor necrosis factor by human cells in vitro and in vivo, induced by group B streptococci.

Tumor necrosis factor alpha (TNF alpha) has been implicated as one of the major mediators of the gram-negative septic shock syndrome. In our studies, group B streptococci (GBS) induced the production of TNF alpha by human mononuclear cells in a dose- and time-dependent manner. Human mixed mononuclear cell cultures exposed to an encapsulated (657.6 +/- 71.3 pg/ml; n = 30 preparations) or an unencapsulated transposon mutant of type III GBS (755.8 +/- 54.7 pg/ml; n = 9) produced similar amounts of TNF alpha. Isolated monocytes and culture-derived macrophages produced higher amounts of TNF alpha (1565 +/- 211 and 1790 +/- 928 pg/ml respectively) in response to GBS than did mixed mononuclear cell cultures. In response to GBS, mixed mononuclear cells from neonates produced significantly more TNF alpha (729.1 +/- 45 vs 520.3 +/- 47.2 pg/ml; p = 0.004) than did cells from adults. Examination of specimens from patients with neonatal GBS disease revealed detectable levels of TNF alpha (7 to 424 pg/ml) in the serum of 5 of 10 patients with sepsis, in 5 of 5 urine samples from infants with sepsis, and in the cerebrospinal fluid of 1 patient with meningitis. These results suggest both a major role for TNF alpha in the pathogenesis of human neonatal GBS sepsis and shock and a potential role for immunotherapy directed against this cytokine in this fulminant neonatal bacterial infection.

Adult↗