Correction: Castanea sativa (European Chestnut) Leaf Extracts Rich in Ursene and Oleanene Derivatives Block Staphylococcus aureus Virulence and Pathogenesis without Detectable Resistance.

[This corrects the article DOI: 10.1371/journal.pone.0136486.].

Staphylococcus aureus Aggregation and Coagulation Mechanisms, and Their Function in Host-Pathogen Interactions.

The human commensal bacterium Staphylococcus aureus can cause a wide range of infections ranging from skin and soft tissue infections to invasive diseases like septicemia, endocarditis, and pneumonia. Muticellular organization almost certainly contributes to S. aureus pathogenesis mechanisms. While there has been considerable focus on biofilm formation and its role in colonizing prosthetic joints and indwelling devices, less attention has been paid to nonsurface-attached group behavior like aggregation and clumping. S. aureus is unique in its ability to coagulate blood, and it also produces multiple fibrinogen-binding proteins that facilitate clumping. Formation of clumps, which are large, tightly packed groups of cells held together by fibrin(ogen), has been demonstrated to be important for S. aureus virulence and immune evasion. Clumps of cells are able to avoid detection by the host's immune system due to a fibrin(ogen) coat that acts as a shield, and the size of the clumps facilitates evasion of phagocytosis. In addition, clumping could be an important early step in establishing infections that involve tight clusters of cells embedded in host matrix proteins, such as soft tissue abscesses and endocarditis. In this review, we discuss clumping mechanisms and regulation, as well as what is known about how clumping contributes to immune evasion.

Evidence for equilibrium iron isotope fractionation by nitrate-reducing iron(II)-oxidizing bacteria.

Iron isotope fractionations produced during chemical and biological Fe(II) oxidation are sensitive to the proportions and nature of dissolved and solid-phase Fe species present, as well as the extent of isotopic exchange between precipitates and aqueous Fe. Iron isotopes therefore potentially constrain the mechanisms and pathways of Fe redox transformations in modern and ancient environments. In the present study, we followed in batch experiments Fe isotope fractionations between Fe(II)(aq) and Fe(III) oxide/hydroxide precipitates produced by the Fe(III) mineral encrusting, nitrate-reducing, Fe(II)-oxidizing Acidovorax sp. strain BoFeN1. Isotopic fractionation in (56)Fe/(54)Fe approached that expected for equilibrium conditions, assuming an equilibrium Δ(56)Fe(Fe(OH)3 - Fe(II)aq) fractionation factor of +3.0 ‰. Previous studies have shown that Fe(II) oxidation by this Acidovorax strain occurs in the periplasm, and we propose that Fe isotope equilibrium is maintained through redox cycling via coupled electron and atom exchange between Fe(II)(aq) and Fe(III) precipitates in the contained environment of the periplasm. In addition to the apparent equilibrium isotopic fractionation, these experiments also record the kinetic effects of initial rapid oxidation, and possible phase transformations of the Fe(III) precipitates. Attainment of Fe isotope equilibrium between Fe(III) oxide/hydroxide precipitates and Fe(II)(aq) by neutrophilic, Fe(II)-oxidizing bacteria or through abiologic Fe(II)(aq) oxidation is generally not expected or observed, because the poor solubility of their metabolic product, i.e. Fe(III), usually leads to rapid precipitation of Fe(III) minerals, and hence expression of a kinetic fractionation upon precipitation; in the absence of redox cycling between Fe(II)(aq) and precipitate, kinetic isotope fractionations are likely to be retained. These results highlight the distinct Fe isotope fractionations that are produced by different pathways of biological and abiological Fe(II) oxidation.

Haematopoietic stem cell recruitment to injured murine liver sinusoids depends on (alpha)4(beta)1 integrin/VCAM-1 interactions.

OBJECTIVE: Evidence suggests haematopoietic stem cells (HSCs) can migrate to injured liver and influence tissue repair. However, mechanisms governing HSC recruitment to injured hepatic microcirculation are poorly understood. These were investigated in vivo following hepatic ischaemia-reperfusion (IR) injury and in vitro using flow-based adhesion assays. DESIGN: Partial IR was induced in anaesthetised WT or PECAM-1(-/-) mice for 90 min. Recruitment of systemically administered HSCs was monitored and effects of function blocking antibodies against alpha(4)beta(1) integrin, CD18, CD44, PECAM-1 or VCAM-1 investigated. The kinetics and molecular events governing adhesion to murine cardiac endothelial cells in vitro were also determined. Effects of conditioned media from IR injured liver on HSC adhesion molecule expression was determined by FACS. RESULTS: Administered HSCs homed predominantly to lungs rather than liver, highlighting a potential therapeutic hurdle. Hepatic HSC recruitment following IR injury was inhibited by anti-alpha(4)beta(1) and anti-VCAM-1 antibodies. A role for alpha(4)beta(1) was also confirmed using flow-based adhesion assays. Incubating HSCs with conditioned media from IR injured liver increased alpha(4)beta(1) expression. CD18, CD44 and PECAM-1 were not involved in recruitment. CONCLUSIONS: This novel study demonstrates that the alpha(4)beta(1)/VCAM-1 pathway mediates HSC recruitment to injured liver. Manipulating this pathway may enhance delivery of HSCs to the liver.

Notch receptor expression in adult human liver: a possible role in bile duct formation and hepatic neovascularization.

Notch signaling is an evolutionarily conserved mechanism, used to regulate cell fate decisions. Four Notch receptors have been identified in man (Notch-1 to -4). In this study, semiquantitative reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry were used to examine the expression pattern of Notch receptor genes in whole adult human liver and isolated liver cell preparations. All 4 receptors were expressed in the adult liver, with no significant differences in the levels of Notch-1, -2, and -4 messenger RNA (mRNA) between normal and diseased liver. However, Notch-3 expression appeared to be increased in diseased tissue. The distribution of Notch-1 and -4 in normal tissue was similar, with Notch-1 also detectable at low levels in the sinusoidal endothelium. Notch-2 expression was more widely distributed, and detectable in hepatocytes, medium-sized bile ducts, and the sinusoidal endothelium. Notch-3 expression was seen on hepatocytes, with weaker expression detectable in portal veins, hepatic arteries, and the sinusoids. In normal liver tissue Notch-1, -2, and -3 were found to be coexpressed on bile duct epithelium; however, with the exception of Notch-3 in primary sclerosing cholangitis (PSC) livers, expression was absent on proliferating ductules in all disease states examined. Interestingly, the expression of Notch-2 and -3 was associated with numerous small vessels within the portal tract septa of diseased tissue. The absence of Notch receptor expression on proliferating bile ductules and its presence on neovessels suggests that Notch signaling may be important for normal bile duct formation and the aberrant neovascularization seen in diseased liver tissue.

CD40 activation-induced, Fas-dependent apoptosis and NF-kappaB/AP-1 signaling in human intrahepatic biliary epithelial cells.

Fas-mediated mechanisms of apoptosis are thought to be involved in the bile duct loss that characterizes diseases such as primary biliary cirrhosis (PBC). We have previously shown that activation of CD40 on hepatocytes can amplify Fas-mediated apoptosis; in the present study, we investigated interactions between CD40 and Fas in biliary epithelial cells (BEC). We report that the bile ducts in PBC liver tissue frequently express increased levels of Fas, Fas ligand (FasL), and CD40 associated with apoptotic BEC. The portal mononuclear infiltrate contains CD40L+ve T cells and macrophages, thereby demonstrating a potential mechanism for CD40 engagement in vivo. Primary cultures of human BEC also expressed Fas, FasL, and CD40 but not CD40L protein or mRNA. Activation of CD40 on BEC using recombinant CD40L increased transcriptional expression of FasL and induced apoptosis, which was inhibited by neutralizing antibodies to either Fas or FasL. Thus, CD40-induced apoptosis of BEC is mediated through Fas/FasL. We then investigated the intracellular signals and transcription factors activated in BEC and found that NF-kappaB and AP-1 were both activated after CD40 ligation. Increased functional NF-kappaB was seen early after CD40 ligation, but returned to baseline levels after 4 h. In contrast, the rapid up-regulation of AP-1 was sustained over 24 h. This study provides further functional evidence of the ability of CD40 to induce Fas/FasL-dependent apoptosis of liver epithelial cells supporting the importance of cross-talk between tumor necrosis factor (TNF) receptor family members as an amplification step in apoptosis induction. Sustained activation of AP-1 in the absence of NF-kappaB signaling may be a critical factor in determining the outcome of CD40 engagement.

Human hepatic stem-like cells isolated using c-kit or CD34 can differentiate into biliary epithelium.

BACKGROUND & AIMS: Recent reports suggest that after bone marrow transplantation into rodents and humans, hematopoietic stem cells migrate into the liver and give rise to oval cells, hepatocytes, and biliary epithelial cells. We investigated this hypothesis further in the human liver using the hematopoietic markers c-kit and CD34. METHODS: Immunofluorescence confocal microscopy was performed using cytokeratin 19 (CK-19; biliary cell marker) with either c-kit or CD34. Immunomagnetic separation was then used to select c-kit- or CD34-positive cells. After attachment, cells were cultured for up to 7 days, and their growth and phenotypic characteristics were examined. RESULTS: In cirrhotic tissue, c-kit- or CD34-positive cells were located in the portal tracts surrounding bile ducts. Occasionally c-kit- (but not CD34-) positive cells that coexpressed CK-19 were observed integrated into bile ducts. In vitro, immunoisolated c-kit or CD34 cells gave rise to colonies of at least 2 morphologies expressing CK-19 or CD31 (endothelial cell marker). CD34- or c-kit-positive cells with similar properties were also isolated from normal liver. CONCLUSIONS: These findings indicate that cells present in human liver that express the markers c-kit or CD34 have the capacity to differentiate into biliary epithelial cell lineage and may therefore represent human biliary epithelial progenitor cells.

Characterization and isolation of ductular cells coexpressing neural cell adhesion molecule and Bcl-2 from primary cholangiopathies and ductal plate malformations.

It has recently been shown that reactive bile ductules display neuroendocrine features, including immunoreactivity for the neural cell adhesion molecule (NCAM). In this study we have compared the immunohistochemical expression of NCAM with that of HEA-125 (biliary specific) and LKM-1 (hepatocyte specific) and other markers relevant to morphogenesis (Bcl-2, EMA) and cell proliferation (Ki-67) in cryostat sections from different chronic liver diseases and from fetal livers at different gestational ages. In parallel, viable NCAM-positive ductular cells were purified from collagenase digests of cirrhotic livers by immunomagnetic separation and characterized by immunocytochemistry and transmission electron microscopy. We demonstrated that reactive ductules with atypical morphology coexpressed NCAM and Bcl-2 and were found mainly in congenital diseases associated with ductal plate malformation and in primary cholangiopathies. On the contrary, reactive ductules with typical morphology were negative for NCAM/Bcl-2 and positive for EMA. Reactive ductules coexpressing NCAM/Bcl-2 were negative for the proliferation marker Ki-67 and appeared to be directly connected with periportal hepatocytes. In fetal livers NCAM/Bcl-2 was transiently expressed during the early developmental stages of ductal plate (10-16 weeks) and started to disappear as the ductal plate began duplicating. NCAM-positive ductal plate cells were Ki-67 negative, becoming positive in duplicated segments. Thus the histogenesis of ductular reactive cells seems to recapitulate the early stages of biliary ontogenesis. In primary cholangiopathies and ductal plate malformations, these cells do not appear to maturate further, and thus abundant ductular structures coexist with vanishing mature ducts. These NCAM-positive ductular cells were immunopurified from patients with chronic cholestatic liver diseases and showed ultrastructural features consistent with a less differentiated phenotype than mature cholangiocytes. These isolated cells represent a useful model for in vitro studies.

Expression of the stem cell factor receptor c-kit in normal and diseased pediatric liver: identification of a human hepatic progenitor cell?

The stem cell factor (SCF)/c-kit ligand/receptor system has been implicated in stem (oval) cell activation following liver injury in the rat. The aim of this study was to determine the role of the SCF/c-kit system in pediatric human liver during acute and chronic liver injury. Tissue was obtained from hepatectomy specimens of patients undergoing liver transplantation for extrahepatic biliary atresia (EHBA) and fulminant hepatic failure (FHF). Specific expression of mRNA for c-kit and beta-actin was measured by ribonuclease protection and by immunohistochemistry to localize c-kit in tissue sections. Expression of c-kit was detected at relatively consistent levels in normal and cirrhotic (EHBA) livers. However, in FHF, c-kit mRNA levels were elevated in 3 of 6 specimens. Immunolocalization highlighted the presence of small numbers of c-kit-positive cells in the portal tracts of normal livers with increased numbers in cirrhotic livers. The highest c-kit staining, however, was observed in FHF, in which, in addition to the cells in the portal tracts, discrete c-kit-positive cells were also found integrated into bile ducts. Colocalization studies demonstrated some of the c-kit-positive cells to be of mast cell, leukocyte, and hematopoietic cell origin. However, there remained a subset that was also negative for these markers. The up-regulation of c-kit receptor expression in diseased livers suggests an involvement of this receptor/ligand system in hepatic repair mechanisms, and we speculate that c-kit-positive cells may represent a hepatic progenitor cell population. The origin and growth/differentiation potential of these c-kit-positive cells is under investigation.

Immunolocalization of OV-6, a putative progenitor cell marker in human fetal and diseased pediatric liver.

The existence of progenitor (stem) cells in the human liver remains a matter of debate. In rodent models of hepatocarcinogenesis and injury, oval cells proliferate in the periportal regions of the portal tracts and are suggested to derive from a stem cell compartment, because they are capable of differentiating into hepatocytes or biliary epithelial cells. In this study, the rat oval cell marker, OV-6 has been used to investigate the hypothesis that there are stem cells present in fetal and pediatric human liver. The pattern of OV-6 expression was compared with the established adult biliary cell markers human epithelial antigen-125 (HEA-125) and cytokeratin-19 (CK-19). In normal pediatric liver (n = 7), bile ducts and ductules were immunostained with CK-19 and HEA-125, whereas OV-6 staining was consistently negative. In fetal tissue (n = 10), ductal plate cells, primitive bile ducts, and hepatoblasts were stained with CK-19 and HEA-125 although only some of the ductal plate cells and hepatoblasts were OV-6 positive. In biliary atresia (n = 6) and 1, anti-trypsin deficiency (1,AT) (n = 4), CK-19 and HEA-125 immunostained ductular proliferative cells that tended to form finely anastomosing ductules, whereas OV-6 staining was found more on discrete cells confined to portal tract margins. Additionally, in diseased liver, OV-6 was strongly positive in hepatocyte lobules with greatest intensity in the periseptal regions. This widespread hepatocyte OV-6 positivity suggests that the antibody may identify cells of a less differentiated phenotype (transitional hepatocytes) that have replaced the mature cells. Therefore, it is proposed that in human liver, OV-6 is recognizing cells with a progenitor stem cell-like phenotype with the capacity to differentiate into OV-6 positive ductular cells or lobular hepatocytes.

Immunolocalization of putative human liver progenitor cells in livers from patients with end-stage primary biliary cirrhosis and sclerosing cholangitis using the monoclonal antibody OV-6.

The term oval cell describes small cells with oval nuclei that arise in the periphery of the portal tracts in rat models of hepatocarcinogenesis and injury and can differentiate into either hepatocytes or bile duct cells, ie, are bipotential. The presence of such cells in human liver is controversial. Here, immunolocalization of OV-6 and two biliary markers, cytokeratin 19 (CK-19) and human epithelial antigen 125 (HEA-125) is compared in normal adult human livers and in primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC) liver sections. CK-19 and HEA-125 stained bile ducts and ductules in normal liver as well as proliferating ductular structures in diseased livers. OV-6 did not label ducts or ductules in normal liver, but in PBC and PSC stained numerous proliferating ductular and periductular cells and lobular hepatocytes. In PBC, discrete OV-6-positive cells with a mature biliary-cell-like morphology were seen integrated into some intact bile ducts as well as occasional small immature oval-like cells. In addition, in PSC, hepatocytes in regenerating lobules were also strongly stained with OV-6, and on close inspection, in both PBC and PSC, oval cells and small hepatocytes at the margins of the lobules were strongly labeled. In contrast to the rat liver, OV-6 and CK-19 staining did not always co-localize. It is proposed that the small OV-6-positive oval cells are analogous to those seen in rat models and may represent human liver progenitor cells that may differentiate into OV-6-positive ductal cells or lobular hepatocytes.

Selective decontamination of the digestive tract reduces gram-negative pulmonary colonization but not systemic endotoxemia in patients undergoing elective liver transplantation.

OBJECTIVE: To examine the effect of selective antibiotic decontamination of the digestive tract in patients undergoing elective orthotopic liver transplantation. DESIGN: Prospective, randomized, concurrent allocation to either selective decontamination or standard antibiotic prophylaxis. SETTING: Operating theater and intensive care unit at a tertiary referral, university teaching hospital. PATIENTS: Fifty-nine adult patients were recruited into the study and underwent liver transplantation. INTERVENTIONS: Thirty-two patients were randomized to standard treatment (control group) and 27 patients were randomized to receive selective decontamination. After early deaths and exclusions, 31 controls and 21 decontamination patients were available for analysis. MEASUREMENTS AND MAIN RESULTS: Portal and systemic endotoxemia, colonization and infection rates, severity of illness (organ system failures, Acute Physiology and Chronic Health Evaluation II score, Therapeutic Intervention Scoring System score), antibiotic costs, and hospital survival rates were measured. Selective decontamination significantly reduced pulmonary infections and enteric, aerobic, and Gram-negative bacillary colonization without facilitating the emergence of resistant organisms, but selective decontamination had no effect on endotoxemia or the development of organ system failures. The financial costs of the selective decontamination regimen outweighed the advantages gained from an associated reduction in antibiotic usage. CONCLUSION: The failure of selective decontamination to enhance survival rates in many studies of the regimen in critically ill patients may, in part, be related to the inability of selective decontamination to abolish endotoxemia.

Antibiotic-induced release of endotoxin from bacteria in vitro.

The ability of cefotaxime, ciprofloxacin, piperacillin and tobramycin to cause release of endotoxin was examined in vitro with cultures of Enterobacter cloacae and Escherichia coli. Endotoxin was measured by a quantitative limulus amoebocyte lysate assay and its presence was confirmed by silver staining of the lipopolysaccharide moiety following SDS-PAGE. The morphology of the bacteria during antibiotic exposure was examined by scanning electronmicroscopy. Cefotaxime, ciprofloxacin and piperacillin caused significant endotoxin release, correlating with their ability to affect cell-wall morphology, causing filamentation, wall breakage and cell lysis. In contrast, little endotoxin was released when bacteria were exposed to tobramycin and no morphological changes were observed when bacteria were exposed to bactericidal concentrations of this aminoglycoside. Its antimicrobial spectrum and bactericidal activity make tobramycin an appropriate agent for treatment of sepsis caused by gram-negative bacteria and its lack of propensity to elicit excessive release of endotoxin may avoid exacerbation of endotoxin-related shock in sepsis.

Analysis of the human serological response to Chlamydia trachomatis 60-kDa proteins by two-dimensional electrophoresis and immunoblotting.

Earlier analyses of sera from humans and animals with chlamydial infections demonstrated a strong immune response to proteins of approximately 60 kDa. We have used two-dimensional (2D) electrophoresis followed by immunoblotting to more accurately define patient responses to the chlamydial 60-kDa cysteine-rich envelope protein (OMP2) and to the 57-kDa stress protein (SP) implicated in immunopathological damage. OMP2 and SP were separated on 2D gels by their distinct isoelectric points and identified by fluorography of [35S]-labelled proteins and cross-reaction with anti-mycobacterial antiserum, respectively. The majority of patients sera showed a strong reaction both to OMP2 and the SP (18/20 and 17/20 sera, respectively). Fewer sera (9/20 and 10/20 sera, respectively) reacted with two other polypeptides also present in the 60-kDa range by this analysis.