Mapping key features of transcriptional regulatory circuitry in embryonic stem cells.

The process by which a single fertilized egg develops into a human being with more than 200 cell types--each with a distinct gene expression pattern controlling its cellular state--is poorly understood. Knowledge of the transcriptional regulatory circuitry that establishes and maintains gene expression programs in mammalian cells is fundamental to understanding development and should provide the foundation for improved diagnosis and treatment of disease. Although it is not yet feasible to map the entirety of this circuitry in vertebrate cells, recent work in embryonic stem (ES) cells has demonstrated that core features of the circuitry can be discovered through studies involving selected regulators. Here, we highlight the fundamental insights that have emerged from studies that examined the role of transcription factors, chromatin regulators, signaling pathways, and noncoding RNAs in the regulatory circuitry of ES cells. Maps of regulatory circuitry and the insights that have emerged from these studies have improved our understanding of global gene expression and are facilitating efforts to reprogram cells for disease therapeutics and regenerative medicine.

Enterobacterial repetitive intergenic consensus polymerase chain reaction typing of isolates of Enterobacter cloacae from an outbreak of infection in a neonatal intensive care unit.

BACKGROUND: Enterobacter cloacae has become a common cause of nosocomial infections. This study was designed to investigate the pattern of spread of E cloacae during an outbreak in a neonatal intensive care unit. METHODS: Enterobacterial repetitive intergenic consensus polymerase chain reaction was used to examine 111 E cloacae isolates from 17 patients, including 81 from surveillance cultures, 23 from endotracheal tubes, 3 from eyes, and 1 each from blood, urine, skin, and throat. Antibiotic susceptibility profiles were also obtained. RESULTS: Infection with E cloacae resulted from endogenous bacteria and from horizontal transmission. One group of 61 isolates, a third of which were obtained from clinical specimens, was uniformly susceptible to imipenem and ciprofloxacin only. A second group of 50 isolates, only 18% of which were obtained from clinical specimens, was susceptible to all antibiotics tested except for aminopenicillins and first-generation cephalosporins. CONCLUSION: These data indicate that (1) patient-to-patient spread is an important cause of E cloacae infection in the neonatal intensive care unit and (2) highly antibiotic-resistant E cloacae may emerge during an outbreak.

Characterization of the mucosal immune response in breast milk after peroral immunization of chimpanzees (Pan troglodytes) with Streptococcus mutans.

The characteristics of the mucosal immune response to Streptococcus mutans cells, antigen A, antigen B, glucosyltransferases and glucan-binding proteins were examined in four pregnant chimpanzees that had been immunized perorally with Strep. mutans. Six pregnant chimpanzees served as non-immunized controls. None of the chimpanzees harbored S. mutans. Samples of milk were collected from all animals throughout the experiment. Peroral immunization resulted in an overall 17-fold median increase in SIgA in milk. Although SIgA1 comprised almost two-thirds of milk SIgA, Strep. mutans whole-cell antibody activity was contained predominantly in the SIgA2 subclass. The difference between the specific activities of anti-Strep. mutans SIgA1 and SIgA2 antibodies compared over time reached the borderline of statistical significance (p = 0.08). The avidity of anti-Strep. mutans antibodies was low in three of four chimpanzees and there was no evidence of affinity maturation. SIgA antibodies from the milk of all four immunized chimpanzees recognized antigen A. In three animals these antibodies were restricted to the SIgA1 subclass and, in one animal, anti-A antibodies were confined to SIgA2. Antibodies from all of the immunized chimpanzees recognized degradation products of antigen B in both the SIgA1 and the SIgA2 subclasses. Only two of four immunized chimpanzees responded to glucosyltransferases and these antibodies were restricted to the SIgA1 subclass. None of the chimpanzees responded to the 74-kDa glucan-binding protein. However, three animals produced SIgA1 antibodies against the 59-kDa glucan-binding protein and two of these also produced SIgA2 antibodies against this protein.

Complement resistance in Borrelia burgdorferi strain 297: outer membrane proteins prevent MAC formation at lysis susceptible sites.

Two variants of Borrelia burgdorferi strain 297, complement-resistant wild-type (WT297) and complement-sensitive mutant (MUT297), were used as a model to study the mechanism of resistance to the alternative complement pathway in this organism. No difference in the quantity of membrane attack complex (MAC) deposition on WT297 and MUT297 was observed after 2 h incubation with normal human serum (NHS), at which time 4% of WT297 and 95% of MUT297 were killed. The polymerization of C9 bound to WT297 and MUT297 was demonstrated by immunoblotting using an anti-C9 polyclonal antibody. Immunofluorescence and thin-section immunoelectron microscopy showed MAC to be diffusely distributed on the outer membrane of both variants. Furthermore, MAC appeared to be tightly bound to the surface of both variants as demonstrated by elution studies. Protease treatment rendered WT297 susceptible to killing by NHS, suggesting that outer membrane proteins may be associated with complement resistance of WT297. One- and two-dimensional gel electrophoreses showed that proteins of 20 and 30 kDa, and 66 kDa were present in WT297 but were absent or sparse in trypsin-treated WT297 and MUT297. Interestingly, immunoblotting using a polyclonal antibody against C3 showed that C3 fragments appeared to bind different acceptors on WT297 than on trypsin-treated WT297, or MUT297. Therefore, the binding of C3 fragments to acceptors on WT297, in contrast to MUT297, may not direct the formation of the MAC to lysis-susceptible sites on the surface of the bacterium, resulting in the complement resistance of WT297.

Humoral immunity to commensal oral bacteria in human infants: salivary secretory immunoglobulin A antibodies reactive with Streptococcus mitis biovar 1, Streptococcus oralis, Streptococcus mutans, and Enterococcus faecalis during the first two years of life.

Secretory immunoglobulin A (SIgA) antibodies reactive with the pioneer oral streptococci Streptococcus mitis biovar 1 and Streptococcus oralis, the late oral colonizer Streptococcus mutans, and the pioneer enteric bacterium Enterococcus faecalis in saliva samples from 10 human infants from birth to age 2 years were analyzed. Low levels of salivary SIgA1 and SIgA2 antibodies reactive with whole cells of all four species were detected within the first month after birth, even though S. mutans and E. faecalis were not recovered from the mouths of the infants during the study period. Although there was a fivefold increase in the concentration of SIgA between birth and age 2 years, there were no differences between the concentrations of SIgA1 and SIgA2 antibodies reactive with the four species over this time period. When the concentrations of SIgA1 and SIgA2 antibodies reactive with all four species were normalized to the concentrations of SIgA1 and SIgA2 in saliva, SIgA1 and SIgA2 antibodies reactive with these bacteria showed a significant decrease from birth to 2 years of age. Adsorption of each infant's saliva with cells of one species produced a dramatic reduction of antibodies recognizing the other three species. Sequential adsorption of saliva samples removed all SIgA antibody to the bacteria, indicating that the SIgA antibodies were directed to antigens shared by all four species. The induction by the host of a limited immune response to common antigens that are likely not involved in adherence may be among the mechanisms that commensal streptococci employ to persist in the oral cavity.

Clonal diversity of vancomycin-resistant enterococci from an outbreak in a tertiary care university hospital.

BACKGROUND: Enterococci have become important nosocomial pathogens and now account for approximately 12% of nosocomial infections. Enterococci can be transferred from patient to patient and from health care personnel to patient. We investigated the clonal diversity of vancomycinresistant enterococci (VRE) causing an outbreak of infections and attempted to determine the patterns of spread of these bacteria in a university hospital. METHODS: Ribotyping was used to examine the clonal diversity of 50 VRE isolates, including 23 from wounds, 14 from urine, 8 from blood, 3 from the rectum, 1 from drainage, and 1 from the cornea. RESULTS: Nine patients were infected with Enterococcus faecalis, 10 with Enterococcus faecium, 3 with both E faecalis and E faecium, and 1 with Enterococcus avium. The results suggest that the sources of the VRE infections included endogenous strains and strains acquired by transmission from attending staff or from the environment. Three patients were infected by both nosocomial and endogenous strains. CONCLUSIONS: These data suggest that the collection and analysis of several isolates from repeated specimens is necessary to obtain a fuller understanding of the epidemiology and population structure of antibiotic-resistant enterococci.

Correlations among Epworth Sleepiness Scale scores, multiple sleep latency tests and psychological symptoms.

The aim of this study was to identify factors other than objective sleep tendency associated with scores on the Epworth Sleepiness Scale (ESS). There were 225 subjects, of whom 40% had obstructive sleep apnoea (OSA), 16% had simple snoring, and 4.9% had snoring with sleep disruption (upper airway resistance syndrome); 9.3% had narcolepsy and 7.5% had hypersomnolence without REM sleep abnormalities; 12% had chronic fatigue syndrome; 7.5% had periodic limb movement disorder and 3% had diurnal rhythm disorders. ESS, the results of overnight polysomnography and multiple sleep latency test (MSLT) and SCL-90 as a measure of psychological symptoms were recorded. The ESS score and the mean sleep latency (MSL) were correlated (Spearman rho = -0.30, P < 0.0001). The MSL was correlated with total sleep time (TST) and with sleep efficiency but not with apnoea/hypopnoea index. There was no association between the MSL and any aspect of SCL-90 scores, except a borderline significant association with the somatisation subscale. The ESS was correlated with TST but not with sleep efficiency or apnoea/hypopnoea index. The ESS was correlated with all subscales of the SCL-90 except psychoticism. An ESS > or = 10 had poor sensitivity and specificity as a predictor of MSL < 10 min or MSL < 5 min. We conclude that the MSLT and the ESS are not interchangeable. The ESS was influenced by psychological factors by which the MSL was not affected. The ESS cannot be used to demonstrate or exclude sleepiness as it is measured by MSLT.

Humoral immunity to commensal oral bacteria in human infants: salivary antibodies reactive with Actinomyces naeslundii genospecies 1 and 2 during colonization.

The secretory immune response in saliva to colonization by Actinomyces naeslundii genospecies 1 and 2 was studied in 10 human infants from birth to 2 years of age. Actinomyces species were not recovered from the mouths of the infants until approximately 4 months after the eruption of teeth. However, low levels of secretory immunoglobulin A1 (SIgA1) and SIgA2 antibodies reactive with whole cells of A. naeslundii genospecies 1 and 2 were detected within the first month after birth. Although there was a fivefold increase in the concentration of SIgA between birth and age 2 years, there were no differences between the concentrations of SIgA1 and SIgA2 antibodies reactive with A. naeslundii genospecies 1 and 2 over this period. When the concentrations of SIgA1 and SIgA2 antibodies reactive with whole cells of A. naeslundii genospecies 1 and 2 were normalized to the concentrations of SIgA1 and SIgA2 in saliva, the A. naeslundii genospecies 1- and 2-reactive SIgA1 and SIgA2 antibodies showed a significant decrease from birth to 2 years of age. The fine specificities of A. naeslundii genospecies 1- and 2-reactive SIgA1 and SIgA2 antibodies were examined by Western blotting of envelope proteins. Similarities in the molecular masses of proteins recognized by SIgA1 and SIgA2 antibodies, both within and between subjects over time, were examined by cluster analysis and showed considerable variability. Taken overall, our data suggest that among the mechanisms Actinomyces species employ to persist in the oral cavity are the induction of a limited immune response and clonal replacement with strains differing in their antigen profiles.

Effect of IgA1 protease on the ability of secretory IgA1 antibodies to inhibit the adherence of Streptococcus mutans.

Secretory IgA (SIgA) is the principal immunoglobulin isotype present in the mucosal secretions of humans. SIgA is thought to play a major role in host defense at these surfaces by inhibiting the colonization of potentially pathogenic microorganisms. A number of bacteria that are mucosal pathogens of humans produce a protease that specifically cleaves the IgA1 subclass of humans and great apes at the hinge region to produce Fab and Fc fragments. In order to study the effect of IgA1 protease on the ability of SIgA1 antibodies to inhibit bacterial adherence, an in vitro assay that quantifies the adsorption of radiolabeled Streptococcus mutans to hydroxyapatite (HA) beads was employed. High titer S. mutans-specific SIgA1 and SIgA2 antibodies were induced in chimpanzee milk for use in the assay. Fab alpha1 fragments had significantly reduced ability to inhibit adherence of S. mutans to saliva-coated HA compared to intact SIgA1 or SIgA2 anti-S. mutans antibodies. These data support the potential importance of IgA1 proteases as an ecological determinant in the oral cavity and their role as a determinant of pathogenesis of pathogenic bacteria whose portal of entry is the mucosal surface.

Increased titre and avidity of IgG antibodies to Porphyromonas gingivalis whole cells and a cell surface protein in subjects with adult periodontitis.

The titre and avidity of IgG antibodies to Porphyromonas gingivalis whole cells and a 47 kDa cell surface protein were determined in serum samples taken from 20 subjects with adult periodontitis and 20 controls, matched for age, gender, ethnic origin and oral hygiene status. Antibody titres were measured by ELISA and antibody avidity was determined by a chaotrope-dissociation ELISA. Avidity was defined as the molarity of chaotrope required to reduce absorbance by 50% (ID50). The mean IgG antibody log titre to whole cells (8.29 vs. 6.92; p < 0.01) and to the 47 kDa antigen (7.61 vs. 6.77; p < 0.05) were higher in cases than in controls. Mean IgG antibody avidity to whole cells (4.59 vs. 2.47; p < 0.001) and to the surface protein (2.54 vs. 1.67; p < 0.001) were also higher in cases than in controls. In cases, IgG antibody titre was highly correlated with avidity for both whole cells (r = 0.878; p = < 0.001) and the 47 kDa protein (r = 0.683; p < 0.001). There was a weaker positive correlation between the titre and the avidity of antibody to whole cells (r = 0.591; p < 0.01) in the control population but antibody titre and avidity for the 47 kDa sonicate antigen were not correlated in the controls (r = 0.104). We conclude that many patients with adult periodontitis have effective humoral immunity to P. gingivalis. However, in up to half the patients with adult periodontitis, antibody titres and avidities were low and similar to control values, indicating either susceptibility due to poor host response or that disease is not associated with this particular pathogen.

Recognition of immunoglobulin A1 by oral actinomyces and streptococcal lectins.

Actinomyces naeslundii and Streptococcus gordonii, oral bacteria that possess Gal/GalNAc- and sialic acid-reactive lectins, respectively, were adherent to immobilized secretory immunoglobulin A (IgA) and two IgA1 myeloma proteins but not to two IgA2 myeloma proteins. Apparently, O-linked oligosaccharides at the hinge region of the IgA1 heavy chain are receptors for lectin-mediated adhesion of these bacteria.

Requirement for the Candida albicans FAS2 gene for infection in a rat model of oropharyngeal candidiasis.

The virulence of Candida albicans strains deficient in fatty acid synthase activity by virtue of disruption/deletion of the FAS2 gene was examined in a rat model of oropharyngeal candidiasis. The FAS2 alleles of C. albicans CAI4 (delta ura3::imm434/delta ura3::imm434) were sequentially disrupted with a cassette that included a portion of FAS2 from which a 984 bp fragment containing the FAS condensing reaction domain was deleted and replaced with hisG-URA3-hisG sequences. Verification of fatty acid synthase inactivation was obtained from assays of enzyme activity. Strains in which a single allele was disrupted (CFD1 and CFD3) exhibited an approximately 20% reduction in activity, when compared to wild-type. In addition, fatty acid synthase activity was abolished in a FAS2 null mutant strain (CFD2), and growth of CFD2 occurred only when the growth medium was supplemented with Tween 40 and certain fatty acids. Strain CFD2 was avirulent in the rat model, indicating that fatty acid synthase activity is required for C. albicans oropharyngeal infection. Strains with a single FAS2 allele disruption colonized the oral cavity, but the number of cells recovered from infected animals was approximately fivefold less than for the parental strain. The results suggest that FAS may be exploited as a possible target for the development of new antifungal agents.

Clonal diversity of Streptococcus mitis biovar 1 isolates from the oral cavity of human neonates.

The clonal diversity of 101 isolates of the pioneer bacterium Streptococcus mitis biovar 1 obtained from the oral cavities of 40 human neonates 1 to 3 days, 2 weeks, and 1 month postpartum was examined by using rRNA gene restriction patterns. There was a high degree of genetic diversity, with the 101 isolates comprising 93 unique PvuII ribotypes. There were eight identical pairs of ribotype patterns, and seven of the eight pairs were obtained from individual neonates. Only one identical pair comprised isolates obtained from different neonates. In all but two cases, isolates with matching ribotypes were obtained at one visit. Two pairs of isolates with matching ribotype patterns were obtained from neonates on successive visits. The ribotype patterns of the isolates were examined by cluster analysis. The isolates forming each cluster were very similar, yet each cluster was well separated from its neighbors. When several isolates were obtained from individual neonates at a particular visit, in some instances they were contained in a single cluster, whereas in other cases each isolate was contained in a separate cluster. Isolates obtained from individual neonates on successive visits tended to be contained in different clusters. This high degree of diversity, which has been observed in other mucosal commensal bacteria, may serve as a mechanism for avoiding immune elimination of these bacteria.

Avirulence of Candida albicans auxotrophic mutants in a rat model of oropharyngeal candidiasis.

The virulence of Candida albicans strain SC5413 and two isogenic derivatives have been investigated in a rat model of oropharyngeal candidiasis. The results demonstrate that both mutant strains are avirulent in this animal model while the parental strain readily initiates infection. Avirulence is not related to altered growth characteristics or the inability of the strains to undergo yeast-to-hyphal morphogenesis. The potential importance of nutritional sufficiency as a virulence factor as well as the possibility of utilizing such strains in the development of an in vitro expression technology system for Candida albicans is discussed.

Identification of pioneer viridans streptococci in the oral cavity of human neonates.

Three hundred and sixty-seven strains of pioneer streptococci isolated from the mouths of 40 healthy, full-term infants during the first month of life were examined by two taxonomic schemes that incorporated biochemical and physiological characteristics, IgA1 protease production and glycosidase activities. Streptococcus mitis biovar 1 and S. oralis comprised 55.0% of the pioneer streptococci isolated from neonates. S. salivarius constituted 25.3% of the isolates, while S. anginosus, S. mitis biovar 2, S. sanguis and S. gordonii accounted collectively for 11.4%. Difficulties in identifying streptococci were encountered and 8.4% of the 367 isolates could not be assigned to a recognised species.

Humoral immunity to commensal oral bacteria: quantitation, specificity and avidity of serum IgG and IgM antibodies reactive with Actinobacillus actinomycetemcomitans in children.

The levels, specificity and avidities of serum IgM and IgG antibodies reactive with Actinobacillus actinomycetemcomitans (Aa) serotypes a, b and c were determined in periodontally healthy (PH) children and compared with subjects with localized juvenile periodontitis (LJP). All PH children exhibited IgM and IgG Aa-reactive antibodies whether or not Aa was detected subgingivally but the antibodies were not specific for Aa. In contrast, LJP sera contained high concentrations of IgM and IgG antibodies reactive with Aa that were largely specific for this bacterium. IgM and IgG antibodies in both PH and LJP subjects were of low avidity. With one exception, the avidities of IgG anti-Aa antibodies were significantly greater than those of IgM antibodies in both PH and LJP subjects. However, although the LJP subjects had as much as 115-fold more Aa-reactive IgG antibody than did the PH subjects the avidities of their IgG antibodies were no greater than those of the PH group. The induction by the host of low-avidity antibodies, that are ineffective in immune elimination, may be a reason why commensal bacteria persist at mucosal surfaces and why persons with LJP fail to eliminate Aa from their periodontal pockets.

Pioneer oral streptococci produce immunoglobulin A1 protease.

As part of a longitudinal study of the relationship between bacterial colonization and the secretory immune response, 367 isolates of pioneer viridans streptococci collected from 40 breast- and bottle-fed neonates within the first month postpartum were tested for the production of immunoglobulin A1 (IgA1) protease and glycosidases. Fifty percent of the streptococci isolated produced IgA1 protease, including all isolates of Streptococcus oralis and S. sanguis, 60.7% of S. mitis biovar 1 isolates, and some isolates that could not be identified. Three cleavage patterns of alpha 1 heavy chains were observed. Six isolates of S. mitis biovar 1 that did not produce IgA1 protease attacked the alpha 1 chain. Incubation of IgA1 protease-negative S. mitis biovar 1 isolates with IgA1, either prior to or together with S. sanguis, rendered the IgA1 paraprotein resistant to cleavage by the IgA1 protease of S. sanguis. The ability of some pioneer streptococci in the human oral cavity to produce IgA1 protease and of others to modify the susceptibility of IgA1 to cleavage by IgA1 protease perhaps enhances their ability to survive in this habitat.

Immunoglobulin A subclasses in infants' saliva and in saliva and milk from their mothers.

We sought to determine (1) the ontogeny of secretory IgA subclasses in saliva of breast- and formula-fed infants and (2) the influence of breast-feeding on the maturation of secretory salivary IgA subclasses. Secretory IgA and subclasses 1 and 2 concentrations were determined in saliva from 40 healthy, term infants from birth to age 18 months, and in parallel milk samples from the infants' mothers who were breast-feeding during the first 6 months after birth. Secretory IgA was detected in the neonates' saliva as early as 3 days after birth, increased rapidly during the next 6 months, but then stabilized at a level approximately one-sixth that of the mothers' salivary secretory IgA. Secretory IgA2 represented less than 15% of secretory IgA in saliva collected 2 weeks after birth but by 6 months represented 24.4% of secretory IgA, a value approaching that of the mothers' salivary secretory IgA2 (30.4%). This increase in the proportion of secretory IgA2 was temporally related to a reduction in the proportion of secretory IgA2 in milk throughout lactation. The secretory IgA concentration increased more rapidly during the first 6 months after birth in infants exclusively breast fed than in those exclusively bottle fed. We conclude that although secretory immunity is immature in infants, breast-feeding may aid in protection against pathogenic microorganisms by increasing the rate of mucosal IgA maturation.

Identification of two subclasses of IgA in the chimpanzee (Pan troglodytes).

Chimpanzee secretory immunoglobulin A (SIgA) was separated into two fractions by chromatography using the terminal galactose-binding lectin Jacalin. The SIgA fraction bound by Jacalin was cleaved by Haemophilus influenzae IgA1 protease, whereas the SIgA nonbinding fraction was not cleaved. It is proposed that these fractions represent IgA1 and IgA2 subclasses because the presence or absence of galactose-terminal oligosaccharides (Jacalin binding) and susceptibility or resistance to IgA1 protease are properties that define human IgA1 and IgA2 subclasses.

Natural transmission of Streptococcus sobrinus in rats: saliva and serum antibody responses to colonization.

One hundred and twenty weanling rats fed diet NIH 2000 that were free of Streptococcus sobrinus and other mutans streptococci were employed in this study. Sixty rats were inoculated orally with S. sobrinus 6715. Each infected rat (donor) was paired and housed with an uninfected recipient. Saliva and serum samples were collected from 24 (12 donor and 12 recipient) rats at the baseline (day 0) and from groups of 12 recipients sacrificed on days 10, 24, 38, and 52, and the level of infection with S. sobrinus was monitored. Salivary immunoglobulin A (IgA) and IgG and serum IgM and IgG antibodies reactive with whole cells (WC), glucosyltransferase (GTF), and the serotype carbohydrate (g) of S. sobrinus were measured by an indirect enzyme-linked immunosorbent assay. Although the rats were free of S. sobrinus and other mutans streptococci at baseline, they exhibited salivary IgA and serum IgM antibodies reactive with S. sobrinus WC, GTF, and g and serum IgG antibodies reactive with WC and GTF. Infection of recipients with S. sobrinus did not induce salivary antibodies reactive with WC, GTF, or g. In contrast, increases in serum IgM and IgG antibodies reactive with WC and serum IgM antibodies reactive with g were observed.