Role of Staphylococcus aureus coagulase and clumping factor in pathogenesis of experimental endocarditis.

The pathogenic role of staphylococcal coagulase and clumping factor was investigated in the rat model of endocarditis. The coagulase-producing and clumping factor-producing parent strain Staphylococcus aureus Newman and a series of mutants defective in either coagulase, clumping factor, or both were tested for their ability (i) to attach in vitro to either rat fibrinogen or platelet-fibrin clots and (ii) to produce endocarditis in rats with catheter-induced aortic vegetations. In vitro, the clumping factor-defective mutants were up to 100 times less able than the wild type strain to attach to fibrinogen and also significantly less adherent than the parents to platelet-fibrin clots. Coagulase-defective mutants, in contrast, were not altered in their in vitro adherence phenotype. The rate of in vivo infection was inoculum dependent. Clumping factor-defective mutants produced ca. 50% less endocarditis than the parent organisms when injected at inoculum sizes infecting, respectively, 40 and 80% (ID40 and ID80, respectively) of rats with the wild-type strain. This was a trend at the ID40 but was statistically significant at the ID80 (P < 0.05). Coagulase-defective bacteria were not affected in their infectivity. Complementation of a clumping factor-defective mutant with a copy of the wild-type clumping factor gene restored both its in vitro adherence and its in vivo infectivity. These results show that clumping factor plays a specific role in the pathogenesis of S. aureus endocarditis. Nevertheless, the rate of endocarditis with clumping factor-defective mutants increased with larger inocula, indicating the contribution of additional pathogenic determinants in the infective process.

Central line sepsis in intensive care units : overview and update

Catheter-related infection remains a leading cause of nosocomial infections, particularly in intensive care units. It includes colonization of the device, skin exit-site infection and device- or catheter-related bloodstream infection. The latter represents the most frequent life-threatening associated complication of central venous catheter use and is associated with significant patient morbidity, mortality and extra hospital costs. The incidence of catheter-related bloodstream infection ranges from 2 to 14 episodes per 1000 catheter-days. On average, microbiologically-documented device-related bloodstream infections complicate from three to five per 100 central venous line uses, but they only represent the visible part of the iceberg and most clinical sepsis are nowadays considered to be catheter-related. We briefly review the pathophysiology of infection, highlighting the importance of the skin insertion site and of intravenous line hub as principal sources of colonization. Principles of therapy are reviewed. Several preventive approaches are also discussed, in particular the possible benefit of recently developed impregnated catheters. Finally, the potential positive impact of a multimodal global preventive strategy based on strict application of hygienic rules is presented.

Citrobacter rodentium Requires Intestinal Neural Wiskott-Aldrich Syndrome Protein (N-WASp) for Actin Pedestal Formation, Colonization and Epithelial Barrier Disruption In Vivo

Background: Citrobacter rodentium is a natural mouse pathogen that is genetically closelyrelated to the human enteric pathogens enteropathogenic and enterohemorrhagic E. coli.Among the repertoire of conserved virulence factors that these pathogens deliver via typeIII secretion, Tir and EspF are responsible for the formation of characteristic actin-richpedestals and disruption of tight junction integrity, respectively. There is evidence In Vitrothese effectors accomplish this, at least in part, by subverting the normal host cellularfunctions of N-WASP, a critical regulator of branched chain actin assembly. Although NWASPhas been shown to be involved in pedestal formation In Vitro, the requirements ofN-WASP-mediated actin pedestals for intestinal colonization by attaching/effacing (A/E)pathogens In Vivo is not known. Furthermore, it is not known whether N-WASP is requiredfor EspF-mediated tight junction disruption. Methods: To investigate the role of N-WASPin the gut epithelium, we generated mice with intestine-specific deletion of N-WASP(iNWKO), by mating mice homozygous for a floxed N-WASP allele (N-WASPL2L/L2L) tomice expressing Cre recombinase under the villin promoter. Separately housed groups ofWT and iNWKO mice were inoculated with 5x108 GFP-expressing C. rodentium by intragastriclavage. Stool was collected 2, 4, 7, and 12 days after infection, and recoverablecolony forming units (CFUs) of C. rodentium were quantified by plating serial dilutions ofhomogenized stool on MacConkey's agar. GFP+ colonies were counted after 24 hoursincubation at 37°C. The presence of actin pedestals was investigated by electron microscopy(EM), and tight junction morphology was assessed by immunofluorescence staining ofoccludin, ZO-1 and claudin-2. Results: C. rodentium infection did not result in mortalityin WT or iNWKO mice. Compared to controls, iNWKO mice exhibited higher levels ofbacterial shedding during the first 4 days of infection (day 4 average: WT 5.2x104 CFU/gvs. iNWKO 4.7x105 CFU/g, p=0.08), followed by a more rapid clearance of C. rodentium, (day7-12 average: WT 2x106 CFU/g vs. iNWKO 2.7x105, p=0.01). EM and immunofluorescencerevealed the complete lack of actin pedestals in iNWKO mice and no mucosa-associatedGFP+ C. rodentium by day 7. WT controls exhibited tight junction disruption, reflected byaltered distribution of ZO-1, whereas iNWKO mice had no change in the pattern of ZO-1.Conclusion: Intestinal N-WASP is required for actin pedestal formation by C. rodentium InVivo, and ablation of N-WASP is associated with more rapid bacterial clearance and decreasedability of C. rodentium to disrupt intercellular junctions.

Hypocretin (orexin) biology and the pathophysiology of narcolepsy with cataplexy.

The discovery of hypocretins (orexins) and their causal implication in narcolepsy is the most important advance in sleep research and sleep medicine since the discovery of rapid eye movement sleep. Narcolepsy with cataplexy is caused by hypocretin deficiency owing to destruction of most of the hypocretin-producing neurons in the hypothalamus. Ablation of hypocretin or hypocretin receptors also leads to narcolepsy phenotypes in animal models. Although the exact mechanism of hypocretin deficiency is unknown, evidence from the past 20 years strongly favours an immune-mediated or autoimmune attack, targeting specifically hypocretin neurons in genetically predisposed individuals. These neurons form an extensive network of projections throughout the brain and show activity linked to motivational behaviours. The hypothesis that a targeted immune-mediated or autoimmune attack causes the specific degeneration of hypocretin neurons arose mainly through the discovery of genetic associations, first with the HLA-DQB1*06:02 allele and then with the T-cell receptor α locus. Guided by these genetic findings and now awaiting experimental testing are models of the possible immune mechanisms by which a specific and localised brain cell population could become targeted by T-cell subsets. Great hopes for the identification of new targets for therapeutic intervention in narcolepsy also reside in the development of patient-derived induced pluripotent stem cell systems.

Bone marrow mesenchymal stem cells stabilize already-formed aortic aneurysms more efficiently than vascular smooth muscle cells in a rat model.

PURPOSE: Abdominal aortic aneurysms (AAAs) expand because of aortic wall destruction. Enrichment in Vascular Smooth Muscle Cells (VSMCs) stabilizes expanding AAAs in rats. Mesenchymal Stem Cells (MSCs) can differentiate into VSMCs. We have tested the hypothesis that bone marrow-derived MSCs (BM-MSCs) stabilizes AAAs in a rat model. MATERIAL AND METHODS: Rat Fischer 344 BM-MSCs were isolated by plastic adhesion and seeded endovascularly in experimental AAAs using xenograft obtained from guinea pig. Culture medium without cells was used as control group. The main criteria was the variation of the aortic diameter at one week and four weeks. We evaluated the impact of cells seeding on inflammatory response by immunohistochemistry combined with RT-PCR on MMP9 and TIMP1 at one week. We evaluated the healing process by immunohistochemistry at 4 weeks. RESULTS: The endovascular seeding of BM-MSCs decreased AAA diameter expansion more powerfully than VSMCs or culture medium infusion (6.5% ± 9.7, 25.5% ± 17.2 and 53.4% ± 14.4; p = .007, respectively). This result was sustained at 4 weeks. BM-MSCs decreased expression of MMP-9 and infiltration by macrophages (4.7 ± 2.3 vs. 14.6 ± 6.4 mm(2) respectively; p = .015), increased Tissue Inhibitor Metallo Proteinase-1 (TIMP-1), compared to culture medium infusion. BM-MSCs induced formation of a neo-aortic tissue rich in SM-alpha active positive cells (22.2 ± 2.7 vs. 115.6 ± 30.4 cells/surface units, p = .007) surrounded by a dense collagen and elastin network covered by luminal endothelial cells. CONCLUSIONS: We have shown in this rat model of AAA that BM-MSCs exert a specialized function in arterial regeneration that transcends that of mature mesenchymal cells. Our observation identifies a population of cells easy to isolate and to expand for therapeutic interventions based on catheter-driven cell therapy.

Advances in paediatric interventional cardiology since 2000.

Interventional paediatric and congenital cardiology is expanding at a rapid pace. Validated techniques (such as aortic or pulmonary valve dilatations and occlusion of persistent ductus arteriosus and atrial septal defects) are improving thanks to the use of smaller introducers and sheaths, low-profile balloons and novel devices. Moreover, catheter-based interventions have emerged as an attractive alternative to surgery in other fields: pulmonary valve replacement, balloon and stent implantation for native and recurrent coarctation, and percutaneous closure of ventricular septal defects. On the other hand, percutaneous interventions in the paediatric population may be limited by patient size or the anatomy of the defect. Hybrid approaches involving both cardiac interventionists and surgeons are being developed to overcome these limitations. Based on a better understanding of cardiac development, fetal cardiac interventions are being attempted in order to alter the history of severe obstructive lesions. Finally, some interventional procedures still carry a low success rate-for example, pulmonary vein stenosis, even with the use of conventional stents. Biodegradable stents and devices are being developed and may find an application in this setting as well as in others. The purpose of this review is to highlight the advances in paediatric interventional cardiology since the beginning of the third millennium.

Pulmonary hypertension complicating isolated atrial septal defect.

Atrial septal defect (ASD) typically is asymptomatic in infancy and early childhood and elective defect closure usually is performed at an age between 4 - 6 years. Severe pulmonary hypertension (PHT) complicating an ASD is seen in adulthood and has only occasionally been reported in small children. A retrospective study was undertaken to evaluate the incidence of severe PHT complicating an isolated ASD and requiring early surgical correction in the fi rst year of life. 355 pediatric patients underwent treatment for an isolated ASD either surgically or by catheter intervention during a 10 year period (1996 - 2006) at two tertiary referral centers. 297 patients had secundum ASD and 58 primum ASD with mild to moderate mitral regurgitation. 8 infants were found with isolated ASD (six with secundum and two with primum ASD) associated with signifi cant PHT, accounting for 2.2% of all ASD patients in our centers. These 8 infants had invasively measured pulmonary artery pressures between 50 and 100% of systemic pressure. Median size of the ASD at the time of surgery was 14mm (7 - 20). They were operated in the fi rst year of life and had complicated postoperative courses requiring specifi c treatment for PHT for up to 16 weeks (median 12) postoperatively. Compared to ASD patients without PHT these infants had prolonged postoperative ICU stay of 5 - 9 days (median 8) and prolonged perioperative overall hospital stay of 8 - 32 days (median 15). Ultimate outcome in all 8 infants was good with persistent normalization of pulmonary pressures during mid-term follow-up of between 8 to 60 months (median 28). All other ASD patients had normal pulmonary pressures and mean age at defect closure was higher being 6.2 years for secundum ASD and 3.2 years for primum ASD. In conclusion, ASD is rarely associated with signifi cant PHT in infancy but then requires early surgery to normalize the prognosis of the patients.

Streptococcus tigurinus is highly virulent in a rat model of experimental endocarditis.

Streptococcus tigurinus is responsible for systemic infections in humans including infective endocarditis. We investigated whether the invasive trait of S. tigurinus in humans correlated with an increased ability to induce IE in rats. Rats with catheter-induced aortic vegetations were inoculated with 10⁴ CFU/ml of either of four S. tigurinus strains AZ_3a(T), AZ_4a, AZ_8 and AZ_14, isolated from patients with infective endocarditis or with the well known IE pathogen Streptococcus gordonii (Challis). Aortic infection was assessed after 24 h. S. tigurinus AZ_3a(T), AZ_4a and AZ_14 produced endocarditis in ≥80% of rats whereas S. gordonii produced endocarditis in only 33% of animals (P<0.05). S. tigurinus AZ_8 caused vegetation infection in 56% of the animals. The capacity of S. tigurinus to induce aortic infection was not related to their ability to bind extracellular matrix proteins (fibrinogen, fibronectin or collagen) or to trigger platelet aggregation. However, all S. tigurinus isolates showed an enhanced resistance to phagocytosis by macrophages and two of them had an increased ability to enter endothelial cells, key attributes of invasive streptococcal species.

Is chemotherapy necessary for premenopausal women with lower-risk node-positive, endocrine responsive breast cancer? 10-year update of International Breast Cancer Study Group Trial 11-93.

INTRODUCTION: International Breast Cancer Study Group (IBCSG) Trial 11-93 is the largest trial evaluating the role of the addition of chemotherapy to ovarian function suppression/ablation (OFS) and tamoxifen in premenopausal patients with endocrine-responsive early breast cancer. METHODS: IBCSG Trial 11-93 is a randomized trial comparing four cycles of adjuvant chemotherapy (AC: doxorubicin or epirubicin, plus cyclophosphamide) added to OFS and 5 years of tamoxifen versus OFS and tamoxifen without chemotherapy in premenopausal patients with node-positive, endocrine-responsive early breast cancer. There were 174 patients randomized from May 1993 to November 1998. The trial was closed before the target accrual was reached due to low accrual rate. RESULTS: Patients randomized tended to have lower risk node-positive disease and the median age was 45. After 10 years median follow up, there remains no difference between the two randomized treatment groups for disease-free (hazard ratio=1.02 (0.57-1.83); P=0.94) or overall survival (hazard ratio=0.97 (0.44-2.16); P=0.94). CONCLUSION: This trial, although small, offers no evidence that AC chemotherapy provides additional disease control for premenopausal patients with lower-risk node-positive endocrine-responsive breast cancer who receive adequate adjuvant endocrine therapy. A large trial is needed to determine whether chemotherapy adds benefit to endocrine therapy for this population.

Frequency of severe pulmonary hypertension complicating "isolated" atrial septal defect in infancy.

Atrial septal defects (ASDs) are typically asymptomatic in infancy and early childhood, and elective defect closure is usually performed at ages of 4 to 6 years. Severe pulmonary hypertension (PH) complicating an ASD is seen in adulthood and has only occasionally been reported in small children. A retrospective study was undertaken to evaluate the incidence of severe PH complicating an isolated ASD and requiring early surgical correction. During a 10-year period (1996 to 2006), 355 pediatric patients underwent treatment for isolated ASDs either surgically or by catheter intervention at 2 tertiary referral centers. Two hundred ninety-seven patients had secundum ASDs, and 58 had primum ASDs with mild to moderate mitral regurgitation. Eight infants were found with isolated ASDs (6 with secundum ASDs and 2 with primum ASDs) associated with significant PH, accounting for 2.2% of all patients with ASDs at the centers. These 8 infants had invasively measured pulmonary artery pressures of 50% to 100% of systemic pressure. They were operated in the first year of life and had complicated postoperative courses requiring specific treatment for PH for up to 16 weeks postoperatively. The ultimate outcomes in all 8 infants were good, with persistent normalization of pulmonary pressures during midterm follow-up of up to 60 months (median 28). All other patients with ASDs had normal pulmonary pressures, and the mean age at defect closure was significantly older, at 6.2 years for secundum ASDs and 3.2 years for primum ASDs. In conclusion, ASDs were rarely associated with significant PH in infancy but then required early surgery and were associated with excellent midterm outcomes in these patients.

Notch1 is required for neuronal and glial differentiation in the cerebellum.

The mechanisms that guide progenitor cell fate and differentiation in the vertebrate central nervous system (CNS) are poorly understood. Gain-of-function experiments suggest that Notch signaling is involved in the early stages of mammalian neurogenesis. On the basis of the expression of Notch1 by putative progenitor cells of the vertebrate CNS, we have addressed directly the role of Notch1 in the development of the mammalian brain. Using conditional gene ablation, we show that loss of Notch1 results in premature onset of neurogenesis by neuroepithelial cells of the midbrain-hindbrain region of the neural tube. Notch1-deficient cells do not complete differentiation but are eliminated by apoptosis, resulting in a reduced number of neurons in the adult cerebellum. We have also analyzed the effects of Notch1 ablation on gliogenesis in vivo. Our results show that Notch1 is required for both neuron and glia formation and modulates the onset of neurogenesis within the cerebellar neuroepithelium.

Qualité de vie après traitement de l'insuffisance veineuse superficielle primaire. [Quality of life in patients traited for primary superficial venous insufficiency].

Venous symptoms and quality of life (QOL) of 78 patients (54 women, mean age 49,5±13,3 years) with primary superficial venous insufficiency (PSVI) were compared at one year after treatment with crossectomy and stripping (C/S, 56 patients) or endovenous laser ablation (EVLA, 22 patients) using the VEINES-QOL questionnaire. Both treatments significantly (p<0,001) improved the scores for venous symptoms (difference 10,6±9,9 and 9,9±8,2 score points for C/S and EVLA, respectively) and QOL (difference 10,3±8,7 and 8,4±6,6 score points for C/S and EVLA, respectively). No difference was found between treatments regarding symptoms or QOL improvement (p=0,30). We conclude that C/S and EVLA are equally effective in improving symptoms and QOL in PSVI.

CD8+ T cell development: CD4 to the rescue.

How immature CD4+CD8+ thymocytes become committed to either the CD4 (helper) or CD8 (cytotoxic) lineage is controversial. Genetic ablation of a silencer element in the gene encoding CD4 provides new evidence that CD8 lineage commitment occurs via a stochastic, rather than instructive, mechanism.

Cardiac raptor deletion impairs adaptive hypertrophy, alters metabolic gene expression and causes heart failure in mice

Background: Mammalian target of rapamycin (mTOR), a central regulator of cell growth, is found in two structurally and functionally distinct multiprotein complexes called mTOR complex (mTORC)1 and mTORC2. The specific roles of each of these branches of mTOR signaling have not been dissected in the adult heart. In the present study, we aimed to bring new insights into the function of cardiac mTORC1-mediated signaling in physiological as well as pathological situations.Methods: We generated mice homozygous for loxP-flanked raptor and positive for the tamoxifen-inducible Cre recombinase (MerCreMer) under control of the α- myosin heavy chain promoter. The raptor gene encodes an essential component of mTORC1. Gene ablation was induced at the age of 10-12 weeks, and two weeks later the raptor cardiac-knockout (raptor-cKO) mice started voluntary cagewheel exercise or were subjected to transverse aortic constriction (TAC) to induce pressure overload.Results: In sedentary raptor-cKO mice, ejection fractions gradually decreased, resulting in significantly reduced values at 38 days (P < 0.001). Raptor-cKO mice started to die during the fifth week after the last tamoxifen injection. At that time, the mortality rate was 36% in sedentary (n = 11) and 64% in exercising (n = 14) mice. TAC-induced pressure overload resulted in severe cardiac dysfunction already at earlier timepoints. Thus, at 7-9 days after surgery, ejection fraction and fractional shortening values were 22.3% vs 43.5% and 10.2% vs 21.5% in raptor-cKO vs wild-type mice, respectively. This was accompanied by significant reductions of ventricular wall and septal thickness as well as an increase in left ventricular internal diameter. Moreover, ventricular weight to tibial length ratios were increased in wild-type, but not in the raptor-cKO TAC mice. Together, this shows that raptor-cKO mice rapidly developed dilated cardiomyopathy without going through a phase of adaptive hypertrophy. Expression of ANP and β-MHC was induced in all raptor-cKO mice irrespective of the cardiac load conditions. Consistent with reduced mTORC1 activity, phosphorylation of ribosomal S6 kinase and 4E-BP1 was blunted, indicating reduced protein synthesis. Moreover, expression of multiple genes involved in the regulation of energy metabolism was altered, and followed by a shift from fatty acid to glucose oxidation.Conclusion: Our study suggests that mTORC1 coordinates protein and energy metabolic pathways in the heart. Moreover, we demonstrate that raptor is essential for the cardiac adaptation to increased workload and importantly, also for normal physiological cardiac function.

In-vivo performance of high-density collagen gel tubes for urethral regeneration in a rabbit model.

Congenital malformations or injuries of the urethra can be treated using existing autologous tissue, but these procedures are sometimes associated with severe complications. Therefore, tissue engineering may be advantageous for generating urethral grafts. We evaluated engineered high-density collagen gel tubes as urethral grafts in 16 male New Zealand white rabbits. The constructs were either acellular or seeded with autologous smooth muscle cells, isolated from an open bladder biopsy. After the formation of a urethral defect by excision, the tissue-engineered grafts were interposed between the remaining urethral ends. No catheter was placed postoperatively. The animals were evaluated at 1 or 3 months by contrast urethrography and histological examination. Comparing the graft caliber to the control urethra at 3 months, a larger caliber was found in the cell-seeded grafts (96.6% of the normal caliber) than in the acellular grafts (42.3%). Histology of acellular and cell-seeded grafts did not show any sign of inflammation, and spontaneous regrowth of urothelium could be demonstrated in all grafts. Urethral fistulae, sometimes associated with stenosis, were observed, which might be prevented by urethral catheter application. High-density collagen gel tubes may be clinically useful as an effective treatment of congenital and acquired urethral pathologies.