ADAM10 mediates vascular injury induced by Staphylococcus aureus α-hemolysin.

Staphylococcus aureus is a leading cause of bacteremia and sepsis. The interaction of S. aureus with the endothelium is central to bloodstream infection pathophysiology yet remains ill-understood. We show herein that staphylococcal α-hemolysin, a pore-forming cytotoxin, is required for full virulence in a murine sepsis model. The α-hemolysin binding to its receptor A-disintegrin and metalloprotease 10 (ADAM10) upregulates the receptor's metalloprotease activity on endothelial cells, causing vascular endothelial-cadherin cleavage and concomitant loss of endothelial barrier function. These cellular injuries and sepsis severity can be mitigated by ADAM10 inhibition. This study therefore provides mechanistic insight into toxin-mediated endothelial injury and suggests new therapeutic approaches for staphylococcal sepsis.

Type I signal peptidase and protein secretion in Staphylococcus epidermidis.

Bacterial protein secretion is a highly orchestrated process that is essential for infection and virulence. Despite extensive efforts to predict or experimentally detect proteins that are secreted, the characterization of the bacterial secretome has remained challenging. A central event in protein secretion is the type I signal peptidase (SPase)-mediated cleavage of the N-terminal signal peptide that targets a protein for secretion via the general secretory pathway, and the arylomycins are a class of natural products that inhibit SPase, suggesting that they may be useful chemical biology tools for characterizing the secretome. Here, using an arylomycin derivative, along with two-dimensional gel electrophoresis and liquid chromatography-tandem mass spectrometry (LC-MS/MS), we identify 11 proteins whose secretion from stationary-phase Staphylococcus epidermidis is dependent on SPase activity, 9 of which are predicted to be translated with canonical N-terminal signal peptides. In addition, we find that the presence of extracellular domains of lipoteichoic acid synthase (LtaS) and the ß-lactam response sensor BlaR1 in the medium is dependent on SPase activity, suggesting that they are cleaved at noncanonical sites within the protein. In all, the data define the proteins whose stationary-phase secretion depends on SPase and also suggest that the arylomycins should be valuable chemical biology tools for the study of protein secretion in a wide variety of different bacteria.

In vitro activities of arylomycin natural-product antibiotics against Staphylococcus epidermidis and other coagulase-negative staphylococci.

The arylomycins are a class of natural-product antibiotics that act via the inhibition of type I signal peptidase (SPase), and we have found in diverse bacteria that their activity is limited by the presence of a resistance-conferring Pro residue in SPase that reduces inhibitor binding. We have also demonstrated that Staphylococcus epidermidis, which lacks this Pro residue, is extremely susceptible to the arylomycins. Here, to further explore the potential utility of the arylomycins, we report an analysis of the activity of a synthetic arylomycin derivative, arylomycin C16, against clinical isolates of S. epidermidis and other coagulase-negative staphylococci (CoNS) from distinct geographical locations. Against many important species of CoNS, including S. epidermidis, S. haemolyticus, S. lugdunensis, and S. hominis, we find that arylomycin C16 exhibits activity equal to or greater than that of vancomycin, the antibiotic most commonly used to treat CoNS infections. While the susceptibility was generally correlated with the absence of the previously identified Pro residue, several cases were identified where additional factors also appear to contribute.

Synthesis and biological evaluation of penem inhibitors of bacterial signal peptidase.

We report the first synthesis of a 5S penem, known to bind bacterial type I signal peptidase, from the commercially available and inexpensive 6-aminopenicillanic acid. We report the first in vivo activity of the compound and use structure-activity relationship studies to begin to define the determinants of signal peptidase binding and also to begin to optimize the penem as an antibiotic.

Growth hormone isoform responses to GABA ingestion at rest and after exercise.

UNLABELLED: Oral administration of the amino acid/inhibitory neurotransmitter gamma aminobutyric acid (GABA) reportedly elevates resting serum growth hormone (GH) concentrations. PURPOSE: To test the hypothesis that GABA ingestion stimulates immunoreactive GH (irGH) and immunofunctional GH (ifGH) release at rest and that GABA augments the resistance exercise-induced irGH/ifGH responses. METHODS: Eleven resistance-trained men (18-30 yr) participated in this randomized, double-blind, placebo-controlled, crossover study. During each experimental bout, participants ingested either 3 g of GABA or sucrose placebo (P), followed either by resting or resistance exercise sessions. Fasting venous blood samples were acquired immediately before and at 15, 30, 45, 60, 75, and 90 min after GABA or P ingestion and were assayed for irGH and ifGH. RESULTS: At rest, GABA ingestion elevated both irGH and ifGH compared with placebo. Specifically, peak concentrations of both hormones were elevated by about 400%, and the area under the curve (AUC) was elevated by about 375% (P < 0.05). Resistance exercise (EX-P) elevated time-point (15-60 min) irGH and ifGH concentrations compared with rest (P < 0.05). The combination of GABA and resistance exercise (EX-GABA) also elevated the peak, AUC, and the 15- to 60-min time-point irGH and ifGH responses compared with resting conditions (P < 0.05). Additionally, 200% greater irGH (P < 0.01) and 175% greater ifGH (P < 0.05) concentrations were observed in the EX-GABA than in the EX-P condition, 30 min after ingestion. GABA ingestion did not alter the irGH to ifGH ratio, and, under all conditions, ifGH represented approximately 50% of irGH. CONCLUSIONS: Our data indicate that ingested GABA elevates resting and postexercise irGH and ifGH concentrations. The extent to which irGH/ifGH secretion contributes to skeletal muscle hypertrophy is unknown, although augmenting the postexercise irGH/ifGH response may improve resistance training-induced muscular adaptations.

High-Throughput Screening Identifies Genes Required for Candida albicans Induction of Macrophage Pyroptosis.

The innate immune system is the first line of defense against invasive fungal infections. As a consequence, many successful fungal pathogens have evolved elegant strategies to interact with host immune cells. For example, Candida albicans undergoes a morphogenetic switch coupled to cell wall remodeling upon phagocytosis by macrophages and then induces macrophage pyroptosis, an inflammatory cell death program. To elucidate the genetic circuitry through which C. albicans orchestrates this host response, we performed the first large-scale analysis of C. albicans interactions with mammalian immune cells. We identified 98 C. albicans genes that enable macrophage pyroptosis without influencing fungal cell morphology in the macrophage, including specific determinants of cell wall biogenesis and the Hog1 signaling cascade. Using these mutated genes, we discovered that defects in the activation of pyroptosis affect immune cell recruitment during infection. Examining host circuitry required for pyroptosis in response to C. albicans infection, we discovered that inflammasome priming and activation can be decoupled. Finally, we observed that apoptosis-associated speck-like protein containing a CARD (ASC) oligomerization can occur prior to phagolysosomal rupture by C. albicans hyphae, demonstrating that phagolysosomal rupture is not the inflammasome activating signal. Taking the data together, this work defines genes that enable fungal cell wall remodeling and activation of macrophage pyroptosis independently of effects on morphogenesis and identifies macrophage signaling components that are required for pyroptosis in response to C. albicans infection.IMPORTANCECandida albicans is a natural member of the human mucosal microbiota that can also cause superficial infections and life-threatening systemic infections, both of which are characterized by inflammation. Host defense relies mainly on the ingestion and destruction of C. albicans by innate immune cells, such as macrophages and neutrophils. Although some C. albicans cells are killed by macrophages, most undergo a morphological change and escape by inducing macrophage pyroptosis. Here, we investigated the C. albicans genes and host factors that promote macrophage pyroptosis in response to intracellular fungi. This work provides a foundation for understanding how host immune cells interact with C. albicans and may lead to effective strategies to modulate inflammation induced by fungal infections.

Acute stimulant ingestion and neurocognitive performance in healthy participants.

CONTEXT: Concussion management has become an area of great concern in athletics, and neurocognitive tests, such as Immediate Post-Concussion Assessment and Cognitive Testing (ImPACT), are commonly used as management tools. Given the restrictive nature of current management plans, anecdotal concerns have been raised about athletes trying to cheat the assessments and return to participation sooner. Stimulants have been shown to improve neurocognitive measures similar to those used in ImPACT. Therefore, they could possibly improve performance during baseline and postinjury testing. OBJECTIVE: To examine the effects of a supplement containing stimulants on ImPACT performance. DESIGN: Crossover study. SETTING: Research laboratory. PATIENTS OR OTHER PARTICIPANTS: A total of 5 men (age = 20.6 ± 1.5 years, height = 176.3 ± 9.6 cm, mass = 76.9 ± 18.6 kg) and 7 women (age = 20.6 ± 1.1 years, height = 162.9 ± 7.8 cm, mass = 60.9 ± 8.2 kg) with no histories of physician-diagnosed head injury, learning disability, or attention-deficit disorder. INTERVENTION(S): Participants were assessed under supplement (5.5 g of Jacked 3D, which contains caffeine and 1,3-dimethylamylamine), placebo, and control conditions separated by 1 week. MAIN OUTCOME MEASURE(S): I compared ImPACT composite scores for verbal and visual memory, visual motor speed, reaction time, impulse control, and a cognitive-efficiency index under each of the 3 conditions and assessed them 30 minutes after ingestion. RESULTS: I observed a difference when comparing reaction times, as the participants reacted faster during the supplement condition (0.53 ± 0.03 seconds) than during the placebo (0.55 ± 0.03 seconds) and control (0.55 ± 0.03 seconds) conditions (F2,22 = 4.31, P = .03). A difference also was observed for the cognitive-efficiency index, as participants scored higher during the supplement condition (0.49 ± 0.09) than during the placebo (0.41 ± 0.10) and control (0.41 ± 0.12) conditions (F2,22 = 4.07, P = .03). CONCLUSIONS: Stimulant ingestion 30 minutes before testing resulted in improved memory, visual processing speed, and reaction time. However, the improvements were relatively nominal, and the question of clinical importance remains. Thus, it is unclear if stimulant ingestion would affect the return-to-participation progression.

Host autophagy combating S. aureus: α-toxin will be tolerated.

Autophagy regulates the degradation of both cellular components and invading intracellular pathogens. In this issue of Cell Host & Microbe, Maurer et al. (2015) reveal that cellular autophagy decreases host sensitivity to Staphylococcus aureus α-toxin via reduced expression of the toxin receptor ADAM10, thus rendering the host tolerant to disease.

Synergistic Action of Staphylococcus aureus α-Toxin on Platelets and Myeloid Lineage Cells Contributes to Lethal Sepsis.

Multi-organ failure contributes to mortality in bacterial sepsis. Platelet and immune cell activation contribute to organ injury during sepsis, but the mechanisms by which bacterial virulence factors initiate these responses remain poorly defined. We demonstrate that during lethal sepsis, Staphylococcus aureus α-toxin simultaneously alters platelet activation and promotes neutrophil inflammatory signaling through interactions with its cellular receptor ADAM10. Platelet intoxication prevents endothelial barrier repair and facilitates formation of injurious platelet-neutrophil aggregates, contributing to lung and liver injury that is mitigated by ADAM10 deletion on platelets and myeloid lineage cells. While platelet- or myeloid-specific ADAM10 knockout does not alter sepsis mortality, double-knockout animals are highly protected. These results define a pathway by which a single bacterial toxin utilizes a widely expressed receptor to coordinate progressive, multi-organ disease in lethal sepsis. As an expression-enhancing ADAM10 polymorphism confers susceptibility to severe human sepsis, these studies highlight the importance of understanding molecular host-microbe interactions.

Assessment of shoulder proprioception in the female softball athlete.

BACKGROUND: There have been reports of overhand throwing athletes having decreased joint position sense in their dominant shoulder as compared with the nondominant shoulder. Very little research, however, exists concerning joint position sense in the female athlete. HYPOTHESIS: Female softball athletes have decreased joint position sense in their dominant shoulder as compared with their nondominant shoulder. STUDY DESIGN: Factorial design with investigation of multiple independent variables. METHODS: Joint position sense was assessed in 50 female softball players and 50 nonthrowing female athletes by using an inclinometer during four glenohumeral joint motions. Both the dominant and nondominant shoulders were assessed and error scores were calculated to describe joint position sense. Data were collected during the course of a fall semester and analyzed by using a mixed model analysis of variance with repeated measures on the dependent variable (error scores). RESULTS: A significant group by movement interaction was observed, with the softball athletes demonstrating significantly greater external rotation error scores than the nonthrowing athletes. CONCLUSION: We failed to reject the null hypothesis. Increased error scores (less joint position sense) were observed in both arms of subjects in the softball group. CLINICAL RELEVANCE: This study suggests that there is decreased shoulder proprioception in asymptomatic female athletes involved in overyhand throwing sports, which may predispose them to injury.

Factor V leiden thrombophilia in a female collegiate soccer athlete: a case report.

OBJECTIVE: To raise awareness among health care providers caring for an active population to an uncommon genetic mutation that increases the risk for a potentially fatal venous thromboembolism. BACKGROUND: A 19-year-old previously healthy female collegiate soccer athlete complained of coughing and progressively decreased exercise tolerance, which were attributed to a recent illness and lack of sleep. Later that evening, she complained of dyspnea and pleuritic pain and was referred to the emergency department. Bilateral pulmonary emboli were identified with computed tomography, and a hypercoagulable panel revealed that the patient was heterozygous for the factor V Leiden mutation. DIFFERENTIAL DIAGNOSIS: Pneumonia, pneumothorax, pericarditis, pleuritis, gastroesophageal reflux disease, pulmonary embolism. TREATMENT: Intravenous heparin therapy was initiated immediately in the emergency department. This was followed by inpatient anticoagulant therapy for 5 days and outpatient anticoagulant therapy for an additional 12 months. During this time, the patient was unable to participate in soccer drills or return to competition and was limited to conditioning activities due to the risk of increased bleeding time. UNIQUENESS: Documented cases of pulmonary embolism in a young athletic population are rare and are usually associated with genetic risk factors. Factor V Leiden is a relatively uncommon genetic mutation that dramatically increases the risk for venous thromboembolism. Although the fatality rate in this population is low, fatality is preventable if the condition is recognized early and managed properly. CONCLUSIONS: Athletes should be encouraged to communicate with their athletic trainers regarding any changes in health status or medication usage. When an athlete presents with nonspecific symptoms such as dyspnea and chest pain, athletic trainers should consider the possibility of pulmonary embolism. A high degree of suspicion results in early diagnosis and treatment and may prevent a fatal event.

Pancreatic laceration in a female collegiate soccer athlete: a case report.

OBJECTIVE: To characterize the diagnosis of pancreatic trauma in an athletic population and to raise awareness among health care providers of the possibility of this life- and organ-threatening injury. BACKGROUND: An 18-year-old, previously healthy female collegiate soccer athlete sustained a direct blow from an opponent's knee between the left and right upper abdominal quadrants while attempting to head the ball. She initially presented with only minimal nausea and discomfort, but this progressed to abdominal pain, tenderness, spasm, and vomiting. She was referred to the emergency department, where she was diagnosed with a pancreatic laceration. DIFFERENTIAL DIAGNOSIS: Duodenal, hepatic, or splenic contusion or laceration; hemorrhagic ovarian cyst. TREATMENT: The patient underwent a distal pancreatectomy and total splenectomy. UNIQUENESS: Pancreatic injuries, particularly those severe enough to warrant surgical intervention, are extremely rare in athletes. CONCLUSIONS: Recognition of a pancreatic injury can be very challenging outside the hospital setting. This is problematic, because a delay in diagnosis is a significant source of preventable morbidity and mortality after this rare injury. Thus, early identification depends on a high index of suspicion, a thorough examination, and close observation. It is imperative that athletic trainers and other health care professionals be able to identify this condition so that referral and management can occur without delay.

National Athletic Trainers' Association position statement: evaluation of dietary supplements for performance nutrition.

OBJECTIVES: To help athletic trainers promote a "food-first" philosophy to support health and performance, understand federal and sport governing body rules and regulations regarding dietary supplements and banned substances, and become familiar with reliable resources for evaluating the safety, purity, and efficacy of dietary supplements. BACKGROUND: The dietary supplement industry is poorly regulated and takes in billions of dollars per year. Uneducated athletes need to gain a better understanding of the safety, eligibility, and efficacy concerns associated with choosing to take dietary supplements. The athletic trainer is a valuable athletic team member who can help in the educational process. In many cases, athletic trainers are asked to help evaluate the legality, safety, and efficacy of dietary supplements. For this position statement, our mission is to provide the athletic trainer with the necessary resources for these tasks. RECOMMENDATIONS: Proper nutrition and changes in the athlete's habitual diet should be considered first when improved performance is the goal. Athletes need to understand the level of regulation (or lack thereof) governing the dietary supplement industry at the international, federal, state, and individual sport-participation levels. Athletes should not assume a product is safe simply because it is marketed over the counter. All products athletes are considering using should be evaluated for purity (ie, truth in labeling), safety, and efficacy.

A Staphylococcus aureus pore-forming toxin subverts the activity of ADAM10 to cause lethal infection in mice.

Staphylococcus aureus is a major cause of human disease, responsible for half a million infections and approximately 20,000 deaths per year in the United States alone. This pathogen secretes α-hemolysin, a pore-forming cytotoxin that contributes to the pathogenesis of pneumonia. α-hemolysin injures epithelial cells in vitro by interacting with its receptor, the zinc-dependent metalloprotease ADAM10 (ref. 6). We show here that mice harboring a conditional disruption of the Adam10 gene in lung epithelium are resistant to lethal pneumonia. Investigation of the molecular mechanism of toxin-receptor function revealed that α-hemolysin upregulates ADAM10 metalloprotease activity in alveolar epithelial cells, resulting in cleavage of the adherens junction protein E-cadherin. Cleavage is associated with disruption of epithelial barrier function, contributing to the pathogenesis of lethal acute lung injury. A metalloprotease inhibitor of ADAM10 prevents E-cadherin cleavage in response to Hla; similarly, toxin-dependent E-cadherin proteolysis and barrier disruption is attenuated in ADAM10-knockout mice. Together, these data attest to the function of ADAM10 as the cellular receptor for α-hemolysin. The observation that α-hemolysin can usurp the metalloprotease activity of its receptor reveals a previously unknown mechanism of pore-forming cytotoxin action in which pathologic insults are not solely the result of irreversible membrane injury and defines ADAM10 inhibition as a strategy to attenuate α-hemolysin-induced disease.

Changes in lower-leg blood flow during warm-, cold-, and contrast-water therapy.

OBJECTIVE: To examine arterial blood flow in the lower leg during warm-, cold-, and contrast-water therapy. DESIGN: A crossover trial with repeated measurements on the dependent variable. SETTING: Hydrotherapy area of a climate-controlled sports medicine clinic. PARTICIPANTS: A volunteer sample of 24 healthy men. INTERVENTION: Four randomly assigned treatments were performed on each subject on consecutive days. MAIN OUTCOME MEASURE: Arterial blood flow (mL per 100mL/min) from baseline measurements were recorded in a 3-minute to 1-minute on-off ratio for 20 minutes by using strain gauge plethysmography. RESULTS: Contrast therapy produced fluctuations in blood flow throughout the 20-minute treatment. Warm-water therapy (40 degrees C) resulted in significant (P < .001) changes in blood flow compared with the control and contrast conditions. Cold-water therapy (13 degrees C) did not produce significantly decreased blood flow as compared with the control condition. CONCLUSIONS: We suggest that further studies involving contrast therapy to the lower leg in injured populations be carried out to determine whether our initial findings are clinically relevant.

The Safety and Efficacy of Anabolic Steroid Precursors: What is the Scientific Evidence?

OBJECTIVE: Anabolic steroid precursors have gained widespread popularity as ergogenic supplements. Advertisements for these supplements claim that they increase endogenous testosterone production and protein synthesis, resulting in increased lean body mass and strength during training. At this time scientific support is limited, but the potential for serious side effects exists and the popularity of these supplements continues to grow. This review provides rationales for the ergogenic claims regarding steroid precursors and compares claims with data from scientifically controlled investigations. DATA SOURCES: A search of MEDLINE and SPORT Discus from 1960 to 2001 using the key words dehydroepiandrosterone, androstenedione, and androstenediol in combination with testosterone, estrogen, exercise, performance, and side effects. DATA SYNTHESIS: Although fairly new to the athletic community, steroid precursors have been used as ergogenic or anabolic agents for quite some time. Suggested gains in strength and lean body mass are attributed to an increase in the endogenous production of testosterone and enhanced protein synthesis. Most of the scientific data, however, do not support manufacturers' ergogenic claims, and the potential for serious side effects, such as decreased high-density lipoprotein cholesterol and increased estrogen concentrations, has been associated with precursor use. Thus, the safety and efficacy of these supplements must be questioned. CONCLUSIONS/RECOMMENDATIONS: It appears that the risks associated with the use of anabolic steroid precursors outweigh any possible ergogenic benefits. Furthermore, these supplements are banned by most athletic organizations. Thus, it is extremely important that athletic trainers are able to educate athletes on these issues so they can continue to perform at an optimum level in a safe and healthy manner.

Cryotherapy does not impair shoulder joint position sense.

OBJECTIVE: To determine the effects of a cryotherapy treatment on shoulder proprioception. DESIGN: Crossover design with repeated measures. SETTING: University athletic training and sports medicine research laboratory. PARTICIPANTS: Thirty healthy subjects (15 women, 15 men). INTERVENTION: A 30-minute cryotherapy treatment. MAIN OUTCOME MEASURES: Joint position sense was measured in the dominant shoulder by using an inclinometer before and after receiving 30 minutes of either no ice or a 1-kg ice bag application. Skin temperature was measured below the tip of the acromion process and recorded every 5 minutes for the entire 30 minutes and immediately after testing. Three different types of error scores were calculated for data analyses and used to determine proprioception. RESULTS: Separate analyses of absolute, constant, and variable error failed to identify changes in shoulder joint proprioception as a function of the cryotherapy application. CONCLUSIONS: Application of an ice bag to the shoulder does not impair joint position sense. The control of proprioception at the shoulder may be more complex than at other joints in the body. Clinical implications may involve modifying rehabilitation considerations when managing shoulder injuries.

Reliability of Joint Position Sense and Force-Reproduction Measures During Internal and External Rotation of the Shoulder.

OBJECTIVE: To determine the reliability of 2 common measures of proprioception. DESIGN AND SETTING: Joint position sense (JPS) and force reproduction (FR) were measured in the dominant shoulder using internal-rotation (IR) and external-rotation (ER) target angles on 2 consecutive days. SUBJECTS: Thirty-one healthy subjects (age = 22.0 +/- 2.8 years, height = 171.8 +/- 9.2 cm, mass = 69.5 +/- 15.9 kg) who did not regularly compete in overhand sports volunteered to participate in the study. MEASUREMENTS: Error scores were calculated at 2 target angles by averaging the absolute difference of 3 trials of JPS and FR. Reliability was determined by comparing the error scores obtained on 2 consecutive days. RESULTS: The inclinometer was found to be a reliable instrument as both intertester (.999) and intratester (.999) intraclass correlation coefficients were high. The JPS and FR measurements were also found to be reliable, with intraclass correlation coefficients ranging from.978 to.984. No differences were observed between trials for either measure. CONCLUSIONS: The inclinometer was a reliable instrument and can provide an affordable and accurate measure of range of motion and JPS. Both JPS and FR were also reliable measures of proprioception in the shoulder. Further research is needed to identify the specific mechanism of proprioception during these tasks.