Modified Bentall operation with bioprosthetic valve and Valsalva graft conduit:the "slit skirt" technique

We elucidated the efficacy of the slit skirt technique to prevent bleeding from the proximal anastomosis between the graft and aortic annulus. Between September 2008 and September 2014, 15 patients underwent a modified Bentall operation with the slit skirt technique at our institution. No patients had bleeding from the proximal anastomosis. No re-thoracotomy for bleeding was required. During midterm follow-up (median period, 21 months), no patient had pseudoaneurysms at the proximal suture line. We conclude that the slit skirt technique is useful to prevent bleeding from the proximal anastomosis after the Bentall operation

Predictor of Early Administration of Antibiotics and a Volume Resuscitation for Young Infants with Septic Shock

(1) Background: It is critical to administer antibiotics and fluid bolus within 1 h of recognizing sepsis in pediatric patients. This study aimed to identify the predictor of the successful completion of a 1-h sepsis bundle for infants with suspected sepsis. (2) Methods: This is an observational study using a prospective registry including febrile young infants (aged < 90 days) who visited a pediatric emergency department with a core body temperature of 38.0 °C or higher and 36.0 °C or lower. Univariate and logistic regression analyses were conducted to determine the predictor (s) of successful sepsis bundle completion. (3) Results: Of the 323 registered patients, 118 patients with suspected sepsis were analyzed, and 38 patients (32.2%) received a bundle-compliant treatment. Among potential variables, such as age, sex, and vital sign parameters, the logistic regression analysis showed that heart rate (odds ratio: OR 1.02; 95% confidence interval: 1.00–1.04) is a significant predictor of the completion of a 1-h sepsis bundle. (4) Conclusions: We found that tachycardia facilitated the sepsis recognition and promoted the successful completion of a 1-h sepsis bundle for young infants with suspected septic shock and a possible indicator for improving the quality of the team-based sepsis management

Limited Utility of SIRS Criteria for Identifying Serious Infections in Febrile Young Infants

(1) Background: Young infants have a high risk of serious infection. The Systematic Inflammatory Response Syndrome (SIRS) criteria can be useful to identify both serious bacterial and viral infections. The aims of this study were to evaluate the diagnostic performance of the SIRS criteria for identifying serious infections in febrile young infants and to identify potential clinical predictors of such infections. (2) Methods: We conducted this prospective cohort study including febrile young infants (aged < 90 days) seen at the emergency department with a body temperature of 38.0 °C or higher. We calculated the diagnostic performance parameters and conducted the logistic regression analysis to identify the predictors of serious infection. (3) Results: Of 311 enrolled patients, 36.7% (n = 114) met the SIRS criteria and 28.6% (n = 89) had a serious infection. The sensitivity, specificity, positive predictive value, and positive likelihood ratio of the SIRS criteria for serious infection was 45.9%, 69.4%, 43.5%, 71.4%, 1.5, and 0.8, respectively. Logistic regression showed that male gender, body temperature ≥ 38.5 °C, heart rate ≥ 178 bpm, and age ≤ 50 days were significant predictors. (4) Conclusions: The performance of the SIRS criteria for predicting serious infections among febrile young infants was poor

Segregation of TRAF6-mediated signaling pathways clarifies its role in osteoclastogenesis

Signals emanating from the receptor for interleukin-1 (IL-1), lipopolysaccharide (LPS) or osteoclast differentiation factor/receptor activator of NFκB ligand (ODF/RANKL) stimulate transcription factors AP-1 through mitogen-activated protein kinase (MAPK) activation and NFκB through IκB kinase (IKK) activation. These kinases are thought to be activated by tumor necrosis factor receptor-associated factor 6 (TRAF6). However, molecular mechanisms by which TRAF6 activates various downstream kinases remain to be elucidated. We identified functional domains of TRAF6 under physiological conditions established by appropriate expression of TRAF6 mutants in TRAF6-deficient cells. In IL-1 and LPS signaling pathways, the RING finger and first zinc finger domains are not required for NFκB activation but are required for full activation of MAPK. However, IL-1 and LPS signals utilize distinct regions within the zinc finger domains of TRAF6 to activate NFκB. Furthermore, the RING finger domain is not required for differentiation of splenocytes to multinuclear osteoclasts, but is essential for osteoclast maturation. Thus, TRAF6 plays essential roles in both the differentiation and maturation of osteoclasts by activating various kinases via its multiple domains

Cell Adhesion Signaling Regulates RANK Expression in Osteoclast Precursors

<div><p>Cells with monocyte/macrophage lineage expressing receptor activator of NF-κB (RANK) differentiate into osteoclasts following stimulation with the RANK ligand (RANKL). Cell adhesion signaling is also required for osteoclast differentiation from precursors. However, details of the mechanism by which cell adhesion signals induce osteoclast differentiation have not been fully elucidated. To investigate the participation of cell adhesion signaling in osteoclast differentiation, mouse bone marrow-derived macrophages (BMMs) were used as osteoclast precursors, and cultured on either plastic cell culture dishes (adherent condition) or the top surface of semisolid methylcellulose gel loaded in culture tubes (non-adherent condition). BMMs cultured under the adherent condition differentiated into osteoclasts in response to RANKL stimulation. However, under the non-adherent condition, the efficiency of osteoclast differentiation was markedly reduced even in the presence of RANKL. These BMMs retained macrophage characteristics including phagocytic function and gene expression profile. Lipopolysaccharide (LPS) and tumor necrosis factor –αTNF-α activated the NF-κB-mediated signaling pathways under both the adherent and non-adherent conditions, while RANKL activated the pathways only under the adherent condition. BMMs highly expressed RANK mRNA and protein under the adherent condition as compared to the non-adherent condition. Also, BMMs transferred from the adherent to non-adherent condition showed downregulated RANK expression within 24 hours. In contrast, transferring those from the non-adherent to adherent condition significantly increased the level of RANK expression. Moreover, interruption of cell adhesion signaling by echistatin, an RGD-containing disintegrin, decreased RANK expression in BMMs, while forced expression of either RANK or TNFR-associated factor 6 (TRAF6) in BMMs induced their differentiation into osteoclasts even under the non-adherent condition. These results suggest that cell adhesion signaling regulates RANK expression in osteoclast precursors.</p> </div

Schema of adherent and non-adherent cell culture systems used in this study.

<p>BMMs (1.25×10⁵/cm²) were cultured on plastic cell culture plates (<i>left</i>: adherent condition) or methylcellulose medium (<i>right</i>: non-adherent condition) in the presence of M-CSF (50 ng/ml), TGF-β (1.0 ng/ml), and RANKL (150 ng/ml) to examine osteoclast differentiation.</p

Osteoclast differentiation induced by forced expression of RANK and TRAF6 in BMMs under adherent and non-adherent condition.

<p>BMMs over-expressing RANK (transduced with pMX-RANK-puro vector) or TRAF6 (transduced with pMX-TRAF6-puro vector) were cultured with M-CSF (50 ng/ml), TGF-β (1 ng/ml), and RANKL (150 ng/ml) for 4 days under the adherent or non-adherent condition, then fixed and stained for TRAP. Cell under non-adherent condition were harvested and placed on cell culture plates for 1 hour to stain for TRAP.</p

Osteoclast differentiation under adherent and non-adherent conditions.

<p><b><i>A</i></b><i>.</i> BMMs were cultured with M-CSF (50 ng/ml), TGF-β (1 ng/ml), and RANKL (150 ng/ml) under the adherent and non-adherent conditions for 24, 48, 72, or 96 hours, after which non-adherent cells were harvested and placed on plastic cell culture plates for 30 minutes, then fixed and stained for TRAP. Cells shown stained red are TRAP-positive cells. <b><i>B</i></b><i>.</i> BMMs grown under adherent and non-adherent conditions were cultured in the presence of M-CSF (50 ng/ml), TGF-β (1 ng/ml), and RANKL (150 ng/ml) for 0, 24, 48, 72, or 96 hours. Subsequently, TRAP activity was determined. Data represent mean values of three independent experiments, with error bars indicating ± SD. <b><i>C</i></b><i>.</i> FITC-conjugated zymosan particles were added to BMMs after 0 and 96 hours of culturing under the adherent and non-adherent conditions in the presence of M-CSF (50 ng/ml), TGF-β (1 ng/ml), and RANKL (150 ng/ml). One hour after zymosan addition, cells were washed with PBS and fixed, then the zymosan particles were visualized by UV illumination. Green dots indicate FITC-conjugated zymosan particles incorporated into cells. <b><i>D</i></b><i>.</i> BMMs were cultured in the presence of M-CSF (50 ng/ml), TGF-β (1 ng/ml), and RANKL (150 ng/ml) under the adherent and non-adherent conditions for 0, 12, 24, 48, and 72 hours, after which mRNA expression levels were examined by RT-PCR. <b><i>E and F</i></b><i>.</i> Relative mRNA expression levels of NFATc1 (<b><i>E</i></b>) and integrin β₃ (<b><i>F</i></b>) at the indicated times under the adherent and non-adherent conditions in the presence of M-CSF (50 ng/ml), TGF-β (1 ng/ml), and RANKL (150 ng/ml), which were quantified using real-time RT-PCR. Data represent the mean values of three independent experiments, with error bars indicating ± SD. **<i>P</i>< 0.01 vs adherent condition at same time point.</p

Effects of RANKL, TNF-α, and LPS on activation of NF-κB pathway.

<p><b><i>A–C</i></b><i>.</i> Cell lysates of BMMs stimulated with RANKL (150 ng/ml) (<i>A</i>), TNF-α (10 ng/ml) (<i>B</i>), or LPS (1 µg/ml) (<b><i>C</i></b>) for the indicated time periods under the adherent and non-adherent conditions were harvested, then phosphorylation and subsequent degradation of IκB were examined by immunoblot analysis. <b><i>D</i></b><i>.</i> BMMs cultured under the adherent and non-adherent conditions were treated with LPS (1 µg/ml) for 3 hours, after which total RNA was extracted and TNF-α mRNA expression levels were examined by RT-PCR.</p

RANK expression levels under adherent and non-adherent conditions.

<p><b><i>A</i></b><i>.</i> BMMs under the adherent condition were harvested (0 h) and subsequently cultured again under adherent and non-adherent conditions in the presence or absence of M-CSF (50 ng/ml), TGF-β (1 ng/ml), and RANKL (150 ng/ml) for the indicated periods. Relative expression levels of RANK mRNA were determined by real time RT-PCR. Data represent mean values of three independent experiments, with error bars indicating ± SD. **<i>P</i>< 0.01 for adherent condition vs. non-adherent condition at the same time point. <b><i>B</i></b><i>. Left panel</i>: BMMs grown under the adherent condition were transferred to the non-adherent condition and further cultured for the indicated time periods. <i>Right panel</i>: BMMs under the non-adherent condition were transferred to the adherent condition and further cultured for the indicated time periods. RANK mRNA expression was analyzed by RT-PCR. <b><i>C</i></b>. <i>Left panel</i>: comparison of RANK protein expression levels between BMMs cultured under the adherent condition and those transferred to the non-adherent condition. <i>Right panel</i>: Comparison of RANK protein levels between BMMs cultured under the non-adherent condition and those transferred to the adherent condition. RANK protein expression on the surface of BMMs was analyzed by flowcytometry.</p