Rapid migration of thymic emigrants to the colonic mucosa in ulcerative colitis patients

Inflammatory bowel disease (IBD) is associated with imbalances of the local intestinal immune responses, with dysregulated CD4⁺ T cells contributing to the chronic inflammation. Having demonstrated altered T cell maturation in the thymus in two different mouse models of colitis, we set out to investigate whether abnormalities in T cell maturation is present in patients with ulcerative colitis (UC) or Crohn's disease (CD). Specimens were obtained from peripheral blood (CD; n = 14, UC; n = 22), colon and small intestinal specimens (CD; n = 6, UC; n = 13). As controls, peripheral blood specimens were obtained from healthy volunteers, patients with adenocarcinomas (n = 18) and colonic specimens from patients with adenocarcinomas (n = 14). Recent thymic emigrants were estimated by analysis of the normalized ratio of T cell receptor excision circles (TRECs) by real‐time polymerase chain reaction (PCR). The frequency of naive‐ and proliferating T lymphocytes and markers of extrathymic T cell maturation in the mucosa was analyzed by flow cytometry and real time‐PCR. TREC levels in peripheral blood T lymphocytes were similar between IBD patients and controls. In contrast, UC patients demonstrated significantly increased levels of TRECs both in intraepithelial and lamina propria lymphocytes from the colonic mucosa compared to patients with adenocarcinomas and CD. However, markers for extrathymic T cell maturation in the mucosa were not different between controls and IBD patients. The increased TREC levels in mucosal but not peripheral blood lymphocytes in UC patients in the absence of increased extrathymic maturation in situ in the mucosa together demonstrate that recent thymic emigrants are recruited rapidly to the inflamed mucosa of these patients.     

Are B lymphocytes of importance in severe Staphylococcus aureus infections?

To investigate the role of B cells in experimental, superantigen-mediated Staphylococcus aureus arthritis and sepsis, we used gene-targeted B-cell-deficient mice. The mice were inoculated intravenously with a toxic shock syndrome toxin 1 (TSST-1)-producing S. aureus strain. The B-cell-deficient and thus agamma-globulinemic mice showed striking similarities to the wild-type control animals with respect to the development of arthritis, the mortality rate, and the rate of bacterial clearance. Surprisingly, we found that the levels of gamma interferon in serum were significantly lower (P < 0. 0001) in B-cell-deficient mice than in the controls, possibly due to impaired superantigen presentation and a diminished expression of costimulatory molecules. In contrast, the levels of interleukin-4 (IL-4), IL-6, and IL-10 in serum were equal in both groups. Our findings demonstrate that neither mature B cells nor their products significantly contribute to the course of S. aureus-induced septic arthritis.     

Immune activation and suppression by group B streptococcus in a murine model of urinary tract infection

Group B streptococcus (GBS) is a common commensal of the gastrointestinal and vaginal mucosa and a leading cause of serious infections in newborns, the elderly, and immunocompromised populations. GBS also causes infections of the urinary tract. However, little is known about host responses to GBS urinary tract infection (UTI) or GBS virulence factors that participate in UTI. Here we describe a novel murine model of GBS UTI that may explain some features of GBS urinary tract association in the human host. We observed high titers and heightened histological signs of inflammation and leukocyte recruitment in the GBS-infected kidney. However, extensive inflammation and leukocyte recruitment were not observed in the bladder, suggesting that GBS may suppress bladder inflammation during cystitis. Acute GBS infection induced the localized expression of proinflammatory cytokines interleukin-1α (IL-1α), macrophage inflammatory protein-1α (MIP-1α), MIP-1β, and IL-9, as well as IL-10, more commonly considered an anti-inflammatory cytokine. Using isogenic GBS strains with different capsule structures, we show that capsular sialic acid residues contribute to GBS urinary tract pathogenesis, while high levels of sialic acid O-acetylation attenuate GBS pathogenesis in the setting of UTI, particularly in direct competition experiments. In vitro studies demonstrated that GBS sialic acids participate in the suppression of murine polymorphonuclear leukocyte (PMN) bactericidal activities, in addition to reducing levels of IL-1α, tumor necrosis factor alpha, IL-1β, MIP-1α, and KC produced by PMNs. These studies define several basic molecular and cellular events characterizing GBS UTI in an animal model, showing that GBS participates simultaneously in the activation and suppression of host immune responses in the urinary tract.     

Population dynamics and niche distribution of uropathogenic Escherichia coli during acute and chronic urinary tract infection

Urinary tract infections (UTIs) have complex dynamics, with uropathogenic Escherichia coli (UPEC), the major causative agent, capable of colonization from the urethra to the kidneys in both extracellular and intracellular niches while also producing chronic persistent infections and frequent recurrent disease. In mouse and human bladders, UPEC invades the superficial epithelium, and some bacteria enter the cytoplasm to rapidly replicate into intracellular bacterial communities (IBCs) comprised of ∼10⁴ bacteria each. Through IBC formation, UPEC expands in numbers while subverting aspects of the innate immune response. Within 12 h of murine bladder infection, half of the bacteria are intracellular, with 3 to 700 IBCs formed. Using mixed infections with green fluorescent protein (GFP) and wild-type (WT) UPEC, we discovered that each IBC is clonally derived from a single bacterium. Genetically tagged UPEC and a multiplex PCR assay were employed to investigate the distribution of UPEC throughout urinary tract niches over time. In the first 24 h postinfection (hpi), the fraction of tags dramatically decreased in the bladder and kidney, while the number of CFU increased. The percentage of tags detected at 6 hpi correlated to the number of IBCs produced, which closely matched a calculated multinomial distribution based on IBC clonality. The fraction of tags remaining thereafter depended on UTI outcome, which ranged from resolution of infection with or without quiescent intracellular reservoirs (QIRs) to the development of chronic cystitis as defined by persistent bacteriuria. Significantly more tags remained in mice that developed chronic cystitis, arguing that during the acute stages of infection, a higher number of IBCs precedes chronic cystitis than precedes QIR formation.     

Lead optimization studies on FimH antagonists: discovery of potent and orally bioavailable ortho-substituted biphenyl mannosides

Herein, we describe the X-ray structure-based design and optimization of biaryl mannoside FimH inhibitors. Diverse modifications to the biaryl ring to improve druglike physical and pharmacokinetic properties of mannosides were assessed for FimH binding affinity based on their effects on hemagglutination and biofilm formation along with direct FimH binding assays. Substitution on the mannoside phenyl ring ortho to the glycosidic bond results in large potency enhancements several-fold higher than those of corresponding unsubstituted matched pairs and can be rationalized from increased hydrophobic interactions with the FimH hydrophobic ridge (Ile13) or "tyrosine gate" (Tyr137 and Tyr48) also lined by Ile52. The lead mannosides have increased metabolic stability and oral bioavailability as determined from in vitro PAMPA predictive model of cellular permeability and in vivo pharmacokinetic studies in mice, thereby representing advanced preclinical candidates with promising potential as novel therapeutics for the clinical treatment and prevention of recurring urinary tract infections.     

Critical review of controversial issues in the management of advanced pediatric liver tumors

Hepatocellular carcinoma (HCC) and hepatoblastoma (HB) are the most common primary tumors of liver in children. The management of patients with locally advanced, unresectable disease or those with extra‐hepatic distant metastases provides substantial challenges to pediatric oncologists, hepatologists, and surgeons. Herein, we critically debate the two sides of three specific controversies: (1) the role of chemotherapy in the treatment of advanced pediatric HCC; (2) the indications for liver transplantation in children with HCC, specifically, the appropriateness of using adult Milan criteria; and (3) the role of liver trasplantation in children with unresectable HB that present with metastatic disease. Pediatr Blood Cancer 2011;56:1013–1018. © 2010 Wiley‐Liss, Inc.     

Contribution of Autolysin and Sortase A during Enterococcus faecalis DNA-Dependent Biofilm Development

Biofilm production is a major attribute of Enterococcus faecalis clinical isolates. Although some factors, such as sortases, autolysin, and extracellular DNA (eDNA), have been associated with E. faecalis biofilm production, the mechanisms underlying the contributions of these factors to this process have not been completely elucidated yet. In this study we define important roles for the major E. faecalis autolysin (Atn), eDNA, and sortase A (SrtA) during the developmental stages of biofilm formation under static and hydrodynamic conditions. Deletion of srtA affects the attachment stage and results in a deficiency in biofilm production. Atn-deficient mutants are delayed in biofilm development due to defects in primary adherence and DNA release, which we show to be particularly important during the accumulative phase for maturation and architectural stability of biofilms. Confocal laser scanning and freeze-dry electron microscopy of biofilms grown under hydrodynamic conditions revealed that E. faecalis produces a DNase I-sensitive fibrous network, which is important for biofilm stability and is absent in atn-deficient mutant biofilms. This study establishes the stage-specific requirements for SrtA and Atn and demonstrates a role for Atn in the pathway leading to DNA release during biofilm development in E. faecalis.Biofilms are bacterial communities encased within an extracellular matrix composed of carbohydrates, proteins, and nucleic acids (10). They are an ideal environment for exchange of genetic materials, such as genes encoding virulence factors and antibiotic resistance determinants, among bacteria within a community (61, 76), while allowing the flow of water and nutrients, as well as ions and various small molecules, to bacteria within the community (8) and providing a protective shield against antibiotics, antimicrobial substances, and phagocytosis (33, 80). Development of a biofilm is a complex multistage process. It is initiated by primary adhesion of the bacteria to a substratum, which is followed by the formation of microcolonies and production of an exopolysaccharide matrix, and it finally culminates with the formation of a three-dimensional (3D) multicellular mature structure (48). Bacterial biofilms are important medically because they have been associated with the pathogenesis of chronic and device-related persistent infections, such as cystic fibrosis, urinary tract infections, and endocarditis (11).Enterococcus faecalis is a gram-positive bacterium that is a commensal of the gastrointestinal tract of healthy individuals. However, it is also an important opportunistic pathogen that is responsible for a wide variety of nosocomial infections, including endocarditis, urinary tract infections, and bacteremia (14, 17, 23, 44). E. faecalis accounts for approximately 65 to 80% of all enterococcal nosocomial infections (16). The ability of E. faecalis to adhere to and develop biofilms on medical devices, such as intravascular and urinary catheters (25, 26, 36, 37, 72), is thought to contribute to its pathogenesis. The increasing resistance of enterococci to most antibiotics, including vancomycin and linezolid (62, 63), especially in biofilms (41, 59, 78), makes treatment of enterococcal infections very difficult.Several putative virulence factors and cellular processes have been implicated in the development of biofilms in E. faecalis (36); however, very little is known about their regulation and molecular contribution to this process. One of these factors, the quorum-sensing two-component transduction signaling system encoded by the fsr locus, an important virulence factor in the pathogenesis of E. faecalis, was shown to control biofilm formation via positive regulation of the extracellular zinc metalloprotease GelE (gelatinase) and the serine protease SprE (19, 38, 54-56). These proteases were recently shown to contribute to enterococcal biofilm formation via regulation of cellular autolysis and fratricidal DNA release (70, 71). In these studies, Thomas et al. also provided the first evidence for a critical role of extracellular DNA (eDNA) in E. faecalis biofilms. eDNA is an important component of the extracellular matrix of bacterial biofilms, providing structural stability to the biofilm and protection against antimicrobials (1, 2, 42, 43, 47, 51, 52, 58, 60, 65, 77). However, the function of this macromolecule throughout the establishment and growth of E. faecalis biofilms is not well characterized.Bacterial murein hydrolases, also referred to as autolysins, have been implicated in biofilm production (58, 60). They are important contributors to cell wall growth and regulation, as well as several lytic processes (75). Two autolysins of Staphylococcus epidermidis, AtlE and Aae, are adhesins that contribute to bacterial attachment to polymeric surfaces and biofilm formation via release of eDNA (20, 21, 58). E. faecalis produces several autolysins, which were recently identified and characterized (12, 24, 35). The major E. faecalis autolysin, Atn (also known as AtlA), is an N-acetylglucosaminidase important for daughter cell separation during cellular division (12). Disruption of atn in E. faecalis resulted in increased chaining, a defect in primary attachment, and decreased biofilm production (3, 24, 31, 37, 57, 70). Recently, Thomas et al. (70) provided evidence that inactivation of this autolysin results in a decrease in DNA release similar to that of gelatinase-deficient mutants. Furthermore, they showed that GelE and SprE can differentially cleave Atn in vitro, and this processing may underlie the mechanism of cell death and DNA release in E. faecalis during biofilm formation.Due to their role in cell wall regulation, autolysins may affect the localization of cell wall-anchored proteins, which can be important for adhesion during biofilm development. In most gram-positive bacteria, membrane-anchored transpeptidase enzymes known as sortases are responsible for covalently anchoring cell surface proteins bearing an LPXTG motif to the cell wall (34). Thus far, only class A and class C sortases have been implicated in biofilm formation by and the virulence of E. faecalis (28). Deletion of srtC, also known as bps, which encodes sortase C (SrtC), resulted in a significant reduction in biofilm production and attenuation of virulence in a mouse model of urinary tract infection, unlike deletion of srtA, which had minor effects under similar conditions (28). However, given the ubiquitous nature of sortases and the limited knowledge of the activity and substrates of the sole SrtA characterized in E. faecalis, it is plausible that this enzyme may play an important role in E. faecalis physiology and/or pathogenesis under different conditions. For instance, SrtA was shown to anchor the plasmid-encoded protein Asc10, which is involved in the pheromone-induced aggregation of E. faecalis (30).In this study, we determined the contributions of E. faecalis SrtA, Atn, and eDNA to the major developmental stages leading to bacterial attachment and the establishment of mature biofilms under static and hydrodynamic conditions. E. faecalis biofilm development occurs in two key steps, which involve primary attachment followed by an accumulative stage. We demonstrate that both SrtA and Atn are required for efficient primary adherence to the substratum, while Atn and eDNA promote high levels of biofilm buildup and architectural stability during the accumulative stage. In the presence of hydrodynamic shear forces, the eDNA in E. faecalis biofilms is associated with a thick and long DNase-sensitive fibrous network that is associated with lysed cells present in the biofilm in some instances, as visualized by freeze-dry microscopy, transmission electron microscopy, and confocal laser scanning microscopy (CLSM). In contrast, atn-deficient mutants are unable to produce visible DNase-sensitive extracellular fibers under these conditions, although the biofilm remains partially sensitive to DNase treatment. These findings argue for a role for Atn in the temporal regulation of DNA release. Collectively, our data underscore the importance of SrtA, the major E. faecalis autolysin, and Atn-mediated DNA release at different stages during the establishment of E. faecalis biofilms. As a critical component of the extracellular matrix of E. faecalis biofilms, eDNA may serve as a novel target for the dissolution of these structures.     

Routine use of a SILASTIC spring-loaded silo for infants with gastroschisis: a multicenter randomized controlled trial

Background

Retrospective studies have suggested that routine use of a preformed silo for infants with gastroschisis may be associated with improved outcomes. We performed a prospective multicenter randomized controlled trial to test this hypothesis.

Methods

Eligible infants were randomized to (1) routine bedside placement of a preformed Silastic spring-loaded silo, with gradual reduction and elective abdominal wall closure, or (2) primary closure.

Results

There were 27 infants in each group. There was no significant difference between groups with respect to age, weight, sex, Apgar scores, prenatal diagnosis, or mode of delivery. The total number of days on the ventilator was lower in the spring-loaded silo group, although it did not reach statistical significance (3.2 vs 5.3, P = .07). There was no significant difference between groups with respect to length of time on total parenteral nutrition, length of stay, or incidence of sepsis and necrotizing enterocolitis.

Conclusion

Routine use of a preformed silo was associated with similar outcomes to primary closure for infants with gastroschisis but with a strong trend toward fewer days on the ventilator. Use of a preformed silo has the advantage of permitting definitive abdominal wall closure in a more elective setting.