Cell-to-cell spread of vaccinia virus is promoted by TGF-ß-independent Smad4 signalling.

The induction of Smad signalling by the extracellular ligand TGF-ß promotes tissue plasticity and cell migration in developmental and pathological contexts. Here, we show that vaccinia virus (VACV) stimulates the activity of Smad transcription factors and expression of TGF-ß/Smad-responsive genes at the transcript and protein levels. Accordingly, infected cells share characteristics to those undergoing TGF-ß/Smad-mediated epithelial-to-mesenchymal transition (EMT). Depletion of the Smad4 protein, a common mediator of TGF-ß signalling, results in an attenuation of viral cell-to-cell spread and reduced motility of infected cells. VACV induction of TGF-ß/Smad-responsive gene expression does not require the TGF-ß ligand or type I and type II TGF-ß receptors, suggesting a novel, non-canonical Smad signalling pathway. Additionally, the spread of ectromelia virus, a related orthopoxvirus that does not activate a TGF-ß/Smad response, is enhanced by the addition of exogenous TGF-ß. Together, our results indicate that VACV orchestrates a TGF-ß-like response via a unique activation mechanism to enhance cell migration and promote virus spread.

A Comparison of the Arterial Blood Concentration of Isoflurane During Cardiopulmonary Bypass Between 2 Polypropylene Oxygenators.

OBJECTIVE: The primary objective was to compare arterial blood concentration of isoflurane during cardiopulmonary bypass (CPB) between 2 polypropylene oxygenators of different designs. Secondary objectives were to compare levels of Bispectral Index Score (BIS) during CPB between the 2 oxygenators and to examine the relationships between oxygenator exhaust and arterial blood concentrations of isoflurane and BIS. DESIGN: Single, blinded, randomized control trial. SETTING: Teaching hospital. PARTICIPANTS: Twenty-five patients undergoing cardiac surgery with CPB. INTERVENTIONS: Subjects were randomly assigned (1:1) to Inspire 8F (Sorin) or Affinity Fusion (Medtronic) oxygenators. MEASUREMENTS AND MAIN RESULTS: The mean arterial blood concentration in the Inspire 8F (Sorin) group was 59 (standard deviation [SD] 23) µg/mL, compared with 53 (SD 17) µg/mL in the Affinity Fusion (Medtronic) group with a nonsignificant mean difference of 6 (95% confidence interval = -11, 22) µg/mL (t[23] = 0.676, p = 0.50). No significant difference in BIS was found between the groups (p = 0.896). Moderate and strong, negative correlations respectively, were found between arterial and oxygenator exhaust correlations and BIS (r = -0.472, p < 0.05; r = -0.812, p < 0.001). A strong, positive correlation was found between arterial and exhaust isoflurane concentration (r = 0.810, p < 0.0005). CONCLUSIONS: No significant difference in arterial blood concentration of isoflurane or BIS was found between the Inspire 8F (Sorin) and Affinity Fusion (Medtronic) oxygenators. A significant positive correlation was found between arterial blood and oxygenator exhaust concentrations of isoflurane, as well as significant negative correlations between both arterial and oxygenator exhaust concentrations of isoflurane and BIS.

Peripheral blood monocyte-derived chemokine blockade prevents murine transfusion-related acute lung injury (TRALI).

Transfusion-related acute lung injury (TRALI) is the leading cause of transfusion-related mortality and can occur with any type of transfusion. TRALI is thought to be primarily mediated by donor antibodies activating recipient neutrophils resulting in pulmonary endothelial damage. Nonetheless, details regarding the interactions between donor antibodies and recipient factors are unknown. A murine antibody-mediated TRALI model was used to elucidate the roles of the F(ab')2 and Fc regions of a TRALI-inducing immunoglobulin G anti-major histocompatibility complex (MHC) class I antibody (34.1.2s). Compared with intact antibody, F(ab')2 fragments significantly increased serum levels of the neutrophil chemoattractant macrophage inflammatory protein 2 (MIP-2); however, pulmonary neutrophil levels were only moderately increased, and no pulmonary edema or mortality occurred. Fc fragments did not modulate any of these parameters. TRALI induction by intact antibody was completely abrogated by in vivo peripheral blood monocyte depletion by gadolinium chloride (GdCl3) or chemokine blockade with a MIP-2 receptor antagonist but was restored upon repletion with purified monocytes. The results suggest a two-step process for antibody-mediated TRALI induction: the first step involves antibody binding its cognate antigen on blood monocytes, which generates MIP-2 chemokine production that is correlated with pulmonary neutrophil recruitment; the second step occurs when antibody-coated monocytes increase Fc-dependent lung damage.

Allogeneic platelet transfusions prevent murine T-cell-mediated immune thrombocytopenia.

Platelet transfusions are life-saving treatments for many patients with thrombocytopenia; however, their use is generally discouraged in the autoimmune disorder known as immune thrombocytopenia (ITP). We examined whether allogeneic platelet major histocompatibility complex (MHC) class I transfusions affected antiplatelet CD61-induced ITP. BALB/c CD61 knockout mice (CD61(-)/H-2(d)) were immunized against platelets from wild-type syngeneic BALB/c (CD61(+)/H-2(d)), allogeneic C57BL/6 (CD61(+)/H-2(b)), or C57BL/6 CD61 KO (CD61(-)/H-2(b)) mice, and their splenocytes were transferred into severe combined immunodeficient (SCID) mice to induce ITP. When nondepleted splenocytes were transferred to induce antibody-mediated ITP, both CD61(+) platelet immunizations generated immunity that caused thrombocytopenia independently of allogeneic MHC molecules. In contrast, when B-cell-depleted splenocytes were transferred to induce T-cell-mediated ITP, transfer of allogeneic MHC-immunized splenocytes completely prevented CD61-induced ITP development. In addition, allogeneic platelet transfusions into SCID mice with established CD61-induced ITP rescued the thrombocytopenia. Compared with thrombocytopenic mice, bone marrow histology in the rescued mice showed normalized megakaryocyte morphology, and in vitro CD61-specific T-cell cytotoxicity was significantly suppressed. These results indicate that antibody-mediated ITP is resistant to allogeneic platelet transfusions, while the T-cell-mediated form of the disease is susceptible, suggesting that transfusion therapy may be beneficial in antibody-negative ITP.

Tracheomalacia Reduces Aerosolized Drug Delivery to the Lung.

Rationale: Neonates with respiratory issues are frequently treated with aerosolized medications to manage lung disease or facilitate airway clearance. Dynamic tracheal collapse (tracheomalacia [TM]) is a common comorbidity in these patients, but it is unknown whether the presence of TM alters the delivery of aerosolized drugs. Objectives: To quantify the effect of neonatal TM on the delivery of aerosolized drugs. Methods: Fourteen infant subjects with respiratory abnormalities were recruited; seven with TM and seven without TM. Respiratory-gated 3D ultrashort echo time magnetic resonance imaging (MRI) was acquired covering the central airway and lungs. For each subject, a computational fluid dynamics simulation modeled the airflow and particle transport in the central airway based on patient-specific airway anatomy, motion, and airflow rates derived from MRI. Results: Less aerosolized drug reached the distal airways in subjects with TM than in subjects without TM: of the total drug delivered, less particle mass passed through the main bronchi in subjects with TM compared with subjects without TM (33% vs. 47%, p = 0.013). In subjects with TM, more inhaled particles were deposited on the surface of the airway (48% vs. 25%, p = 0.003). This effect becomes greater with larger particle sizes and is significant for particles with a diameter >2 µm (2-5 µm, p ≤ 0.025 and 5-15 µm, p = 0.004). Conclusions: Neonatal patients with TM receive less aerosolized drug delivered to the lungs than subjects without TM. Currently, infants with lung disease and TM may not be receiving adequate and/or expected medication. Particles >2 µm in diameter are likely to deposit on the surface of the airway due to anatomical constrictions such as reduced tracheal and glottal cross-sectional area in neonates with TM. This problem could be alleviated by delivering smaller aerosolized particles.

Cellular immune dysfunction in immune thrombocytopenia (ITP).

Over the past decades, a wealth of information has been reported about the pathogenic features of immune thrombocytopenia (ITP). To this day, however, it is unclear whether the immune abnormalities associated with ITP play causative roles in the disease or are secondary epiphenomena brought on by the inflammatory processes that are associated with the disorder. Like the majority of all autoimmune diseases, ITP is an organ-specific disease and abnormalities in immune cell types, such as antigen-presenting cells (APC), T cells and B cells have been shown to play some sort of role in the initiation and/or perpetuation of the disease. This review will discuss recent advances in understanding three immune cells important in ITP pathophysiology: APC, T cells and B cells, and will review how they interact with each other to initiate and perpetuate ITP, particularly the chronic form of the disorder. It will also focus on new data related to the genetics of the disorder and discuss relevant animal models of ITP.

The evolving classifications and epidemiological challenges surrounding chronic migraine and medication overuse headache: a review.

Changes in diagnostic criteria, for example, the various International Classification of Headache Disorders criteria, would lead to changes in the outcomes of epidemiological studies. International Classification of Headache Disorders-1 was based mainly on expert opinion, yet most of the diagnostic criteria were reliable and valid, but it did not include chronic migraine. In its second version, the classification introduced chronic migraine, but this diagnosis resembled more a high-frequency migraine rather than the actual migraine transformation process. It also introduced medication overuse headache, but it necessitated analgesic withdrawal and subsequent headache improvement to be diagnosed as such. Hence patients having medication overuse headache could only be diagnosed in retrospect, which was an awkward situation. Such restrictive criteria for chronic migraine and medication overuse headache omitted a high proportion of patients. International Classification of Headache Disorders-3 allows a diagnosis of medication overuse headache due to combination analgesics if taken for at least 10 days per month for more than three months. Hence the prevalence rate of medication overuse headache and chronic migraine can increase compared to the previous version of the headache classification. Different criteria have been used across studies to identify chronic migraine and medication overuse headache, and therefore the information acquired from previous studies using earlier criteria becomes uncertain. Hence much epidemiological research would need to be interpreted cautiously or repeated with the most updated criteria, since the subjects in studies that apply the latest criteria may be phenotypically different from those in older studies.

Insertion sequence-driven evolution of Escherichia coli in chemostats.

Insertion sequence (IS) elements are present in almost all bacterial genomes and are mobile enough to provide genomic tools to differentiate closely related isolates. They can be used to estimate genetic diversity and identify fitness-enhancing mutations during evolution experiments. Here, we determined the genomic distribution of eight IS elements in 120 genomes sampled from Escherichia coli populations that evolved in glucose- and phosphate-limited chemostats by comparison to the ancestral pattern. No significant differential transposition of the various IS types was detected across the environments. The phylogenies revealed substantial diversity amongst clones sampled from each chemostat, consistent with the phenotypic diversity within populations. Two IS-related changes were common to independent chemostats, suggesting parallel evolution. One of them corresponded to insertions of IS1 elements within rpoS encoding the master regulator of stress conditions. The other parallel event was an IS5-dependent deletion including mutY involved in DNA repair, thereby providing the molecular mechanism of generation of mutator clones in these evolving populations. These deletions occurred in different co-existing genotypes within single populations and were of various sizes. Moreover, differential locations of IS elements combined with their transpositional activity provided evolved clones with different phenotypic landscapes. Therefore, IS elements strongly influenced the evolutionary processes in continuous E. coli cultures by providing a way to modify both the global regulatory network and the mutation rates of evolving cells.

Lumbopelvic control and pitching performance of professional baseball pitchers.

This study assessed the correlation between lumbopelvic control during a single-leg balancing task and in-game pitching performance in Minor-League baseball pitchers. Seventy-five healthy professional baseball pitchers performed a standing lumbopelvic control test during the last week of spring training for the 2008 and 2009 seasons while wearing a custom-designed testing apparatus, the "Level Belt." With the Level Belt secured to the waist, subjects attempted to transition from a 2-leg to a single-leg pitching stance and balance while maintaining a stable pelvic position. Subjects were graded on the maximum sagittal pelvic tilt from a neutral position during the motion. Pitching performance, number of innings pitched (IP), and injuries were compared for all subjects who pitched at least 50 innings during a season. The median Level Belt score for the study group was 7°. Two-sample t-tests with equal variances were used to determine if pitchers with a Level Belt score <7° or ≥7° were more likely to perform differently during the baseball season, and chi-square analysis was used to compare injuries between groups. Subjects scoring <7° on the Level Belt test had significantly fewer walks plus hits per inning than subjects scoring ≥7° (walks plus hits per inning pitched, 1.352 ± 0.251 vs. 1.584 ± 0.360, p = 0.013) and significantly more IP during the season (IP, 78.89 ± 38.67 vs. 53.38 ± 42.47, p = 0.043). There was no significant difference in the number of pitchers injured between groups. These data suggest that lumbopelvic control influences overall performance for baseball pitchers and that a simple test of lumbopelvic control can potentially identify individuals who have a better chance of pitching success.

Genomic identification of a novel mutation in hfq that provides multiple benefits in evolving glucose-limited populations of Escherichia coli.

Beneficial mutations in diversifying glucose-limited Escherichia coli populations are mostly unidentified. The genome of an evolved isolate with multiple differences from that of the ancestor was fully assembled. Remarkably, a single mutation in hfq was responsible for the multiple benefits under glucose limitation through changes in at least five regulation targets.

Divergent roles of ß- and γ-actin isoforms during spread of vaccinia virus.

Actin is a major component of the cytoskeleton and is present as two isoforms in non-muscle cells: ß- and γ-cytoplasmic actin. These isoforms are strikingly conserved, differing by only four N-terminal amino acids. During spread from infected cells, vaccinia virus (VACV) particles induce localized actin nucleation that propel virus to surrounding cells and facilitate cell-to-cell spread of infection. Here we show that virus-tipped actin comets are composed of ß- and γ-actin. We employed isoform-specific siRNA knockdown to examine the role of the two isoforms in VACV-induced actin comets. Despite the high level of similarity between the actin isoforms, and their colocalization, VACV-induced actin nucleation was dependent exclusively on ß-actin. Knockdown of ß-actin led to a reduction in the release of virus from infected cells, a phenotype dependent on virus-induced Arp2/3 complex activity. We suggest that local concentrations of actin isoforms may regulate the activity of cellular actin nucleator complexes.

Congenital Tuberculosis Complicated by Hemophagocytic Lymphohistiocytosis.

We present the case of a male infant with congenital tuberculosis in a nonendemic setting complicated by hemophagocytic lymphohistiocytosis, who was treated successfully with antituberculous therapy and corticosteroids. We review the pediatric literature concerning the unusual association of these 2 rare conditions.

Anterior Cruciate Ligament Reconstruction Rehabilitation: MOON Guidelines.

CONTEXT: Anterior cruciate ligament (ACL) reconstruction rehabilitation has evolved over the past 20 years. This evolution has been driven by a variety of level 1 and level 2 studies. EVIDENCE ACQUISITION: The MOON Group is a collection of orthopaedic surgeons who have developed a prospective longitudinal cohort of the ACL reconstruction patients. To standardize the management of these patients, we developed, in conjunction with our physical therapy committee, an evidence-based rehabilitation guideline. STUDY DESIGN: Clinical review. LEVEL OF EVIDENCE: Level 2. RESULTS: This review was based on 2 systematic reviews of level 1 and level 2 studies. Recently, the guideline was updated by a new review. Continuous passive motion did not improve ultimate motion. Early weightbearing decreases patellofemoral pain. Postoperative rehabilitative bracing did not improve swelling, pain range of motion, or safety. Open chain quadriceps activity can begin at 6 weeks. CONCLUSION: High-level evidence exists to determine appropriate ACL rehabilitation guidelines. Utilizing this protocol follows the best available evidence.

Lumbopelvic control and days missed because of injury in professional baseball pitchers.

BACKGROUND: Recently, lumbopelvic control has been linked to pitching performance, kinematics, and loading; however, poor lumbopelvic control has not been prospectively investigated as a risk factor for injuries in baseball pitchers. HYPOTHESIS: Pitchers with poor lumbopelvic control during spring training are more likely to miss ≥30 days because of an injury through an entire baseball season than pitchers with good lumbopelvic control. STUDY DESIGN: Cohort study; Level of evidence, 2. METHODS: A total of 347 professional baseball pitchers were enrolled into the study during the last 2 weeks of spring training and stayed with the same team for the entire season. Lumbopelvic control was quantified by peak anterior-posterior deviation of the pelvis relative to the starting position during a single-leg raise test (APScore). Days missed because of an injury through the entire season were recorded by each team's medical staff. RESULTS: A higher APScore was significantly associated with a higher likelihood of missing ≥30 days (P = .023, χ(2) test). When divided into tertiles based on their APScore, participants in the highest tertile were 3.0 times and 2.2 times more likely to miss at least 30 days throughout the course of a baseball season relative to those in the lowest or middle tertiles, respectively. A higher APScore was also significantly associated with missing more days because of an injury within participants who missed at least 1 day (P = .018, ANOVA), with participants in the highest tertile missing significantly more days (mean, 98.6 days) than those in the middle tertile (mean, 45.8 days; P = .017) or lowest tertile (mean, 43.8 days; P = .017). CONCLUSION: This study found that poor lumbopelvic control in professional pitchers was associated with an increased risk of missing significant time because of an injury.

Ybp1 and Gpx3 signaling in Candida albicans govern hydrogen peroxide-induced oxidation of the Cap1 transcription factor and macrophage escape.

AIMS: As Candida albicans is the major fungal pathogen of humans, there is an urgent need to understand how this pathogen evades toxic reactive oxygen species (ROS) generated by the host immune system. A key regulator of antioxidant gene expression, and thus ROS resistance, in C. albicans is the AP-1-like transcription factor Cap1. Despite this, little is known regarding the intracellular signaling mechanisms that underlie the oxidation and activation of Cap1. Therefore, the aims of this study were; (i) to identify the regulatory proteins that govern Cap1 oxidation, and (ii) to investigate the importance of Cap1 oxidation in C. albicans pathogenesis. RESULTS: In response to hydrogen peroxide (H2O2), but not glutathione-depleting/modifying oxidants, Cap1 oxidation, nuclear accumulation, phosphorylation, and Cap1-dependent gene expression, is mediated by a glutathione peroxidase-like enzyme, which we name Gpx3, and an orthologue of the Saccharomyces cerevisiae Yap1 binding protein, Ybp1. In addition, Ybp1 also functions to stabilise Cap1 and this novel function is conserved in S. cerevisiae. C. albicans cells lacking Cap1, Ybp1, or Gpx3, are unable to filament and thus, escape from murine macrophages after phagocytosis, and also display defective virulence in the Galleria mellonella infection model. INNOVATION: Ybp1 is required to promote the stability of fungal AP-1-like transcription factors, and Ybp1 and Gpx3 mediated Cap1-dependent oxidative stress responses are essential for the effective killing of macrophages by C. albicans. CONCLUSION: Activation of Cap1, specifically by H2O2, is a prerequisite for the subsequent filamentation and escape of this fungal pathogen from the macrophage.

Interactions between macrophages and cell wall oligosaccharides of Candida albicans.

The fungal cell wall is the armour that protects the cell from changes in the external environment. The wall of Candida albicans, an opportunistic human pathogen, is also the immediate point of contact with the host immune system and contains most of the pathogen-associated molecular patterns recognised by innate immune cells. Along with the use of mutants altered in cell wall composition, the isolation and purification of cell wall components has proven useful in the identification of receptors involved in the sensing of these molecules, and assessment of the relative relevance of ligand-receptor interactions during the sensing of C. albicans by the immune system. Here, we describe protocols for the isolation of cell wall chitin, N-linked and O-linked mannans from C. albicans, and how they can subsequently be used to assess immunological activities such as phagocytosis and cytokine production by myeloid cells.

Divergence involving global regulatory gene mutations in an Escherichia coli population evolving under phosphate limitation.

Many of the important changes in evolution are regulatory in nature. Sequenced bacterial genomes point to flexibility in regulatory circuits but we do not know how regulation is remodeled in evolving bacteria. Here, we study the regulatory changes that emerge in populations evolving under controlled conditions during experimental evolution of Escherichia coli in a phosphate-limited chemostat culture. Genomes were sequenced from five clones with different combinations of phenotypic properties that coexisted in a population after 37 days. Each of the distinct isolates contained a different mutation in 1 of 3 highly pleiotropic regulatory genes (hfq, spoT, or rpoS). The mutations resulted in dissimilar proteomic changes, consistent with the documented effects of hfq, spoT, and rpoS mutations. The different mutations do share a common benefit, however, in that the mutations each redirect cellular resources away from stress responses that are redundant in a constant selection environment. The hfq mutation lowers several individual stress responses as well the small RNA-dependent activation of rpoS translation and hence general stress resistance. The spoT mutation reduces ppGpp levels, decreasing the stringent response as well as rpoS expression. The mutations in and upstream of rpoS resulted in partial or complete loss of general stress resistance. Our observations suggest that the degeneracy at the core of bacterial stress regulation provides alternative solutions to a common evolutionary challenge. These results can explain phenotypic divergence in a constant environment and also how evolutionary jumps and adaptive radiations involve altered gene regulation.