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The Wnt/β-catenin signaling pathway has been implicated in human proliferative diseases such as cancer and fibrosis. The functions of β-catenin and several other components of this pathway have been investigated in fibrosis. However, the potential role of R-spondin proteins (RSPOs), enhancers of the Wnt/β-catenin signaling, has not been described. A specific interventional strategy targeting this pathway for fibrosis remains to be defined. We developed monoclonal antibodies against members of the RSPO family (RSPO1, 2, and 3) and probed their potential function in fibrosis in vivo. We demonstrated that RSPO3 plays a critical role in the development of fibrosis in multiple organs. Specifically, an anti-RSPO3 antibody, OMP-131R10, when dosed therapeutically, attenuated fibrosis in carbon tetrachloride (CCl4)-induced liver fibrosis, bleomycin-induced pulmonary and skin fibrosis models. Mechanistically, we showed that RSPO3 induces multiple pro-fibrotic chemokines and cytokines in Kupffer cells and hepatocytes. We found that the anti-fibrotic activity of OMP-131R10 is associated with its inhibition of β-catenin activation in vivo. Finally, RSPO3 was found to be highly elevated in the active lesions of fibrotic tissues in mouse models of fibrosis and in patients with idiopathic pulmonary fibrosis (IPF) and nonalcoholic steatohepatitis (NASH). Together these data provide an anti-fibrotic strategy for targeting the Wnt/β-catenin pathway through RSPO3 blockade and support that OMP-131R10 could be an important therapeutic agent for fibrosis.

Purpose: Lower extremity chronic exertional compartment syndrome (LE-CECS) can limit rigorous physical activity, particularly in the US military, and fasciotomy is a potential treatment. Associations between LE-CECS, fasciotomy, and occupational outcomes appear understudied. We studied active-duty US service members to identify whether LE-CECS diagnoses and fasciotomy for LE-CECS predict military service separation., Methods: We conducted a retrospective cohort study of 1,103,417 individuals who entered service during 2011 to 2017. Distributions of separation statuses and independent variables were tabulated, and sex-specific multivariable regression models were computed for separation outcomes., Results: LE-CECS was associated with a 474% medical service separation risk in men (95% confidence interval (CI) for adjusted risk ratio: 5.21 to 6.33) and a 282% increase in women (CI: 2.99 to 4.88). Among 1947 patients with LE-CECS, men saw a 57% nonmedical separation risk increase (CI: 1.27 to 1.93) and women had a 119% increase (CI: 1.10 to 4.35) when anterior and/or lateral compartment procedures occurred. Men with LE-CECS and any posterior procedures saw a 47% nonmedical separation risk increase (CI: 1.17 to 1.86). Men with anterior and/or lateral procedures and any posterior procedures experienced 36% (CI: 1.09 to 1.71) and 78% (CI: 1.40 to 2.26) medical separation risk increases, respectively. No statistically significant risk increases for the outcomes were otherwise seen for women., Conclusions: LE-CECS was associated with increased military service discharge risks. Among the affected patients, positive effects on career longevity were not seen in association with fasciotomy. However, fasciotomy could simply serve as a marker of the most refractory cases that are least likely to continue service. More study is needed to clarify causal pathways and identify patients who may derive career benefits from surgical treatment., (Copyright © 2024 by the American College of Sports Medicine.)

Background: Overdose rates in rural areas have been increasing globally, with large increases in the United States. Few studies, however, have identified correlates of non-fatal overdose among rural people who use drugs (PWUD). The present analysis describes correlates of nonfatal overdose among a large multistate sample of rural PWUD., Methods: This is a cross-sectional analysis of data gathered via surveys with PWUD recruited through seven Rural Opioid Initiative (ROI) sites. Descriptive analyses were conducted to assess the prevalence of past 30-day overdose. Generalized estimating equations were used to estimate a series of multivariable models quantifying relationships of select factors to past-month overdose; factors were selected using the Risk Environment Framework., Results: The multisite sample included 2711 PWUD, 6% of whom reported overdosing in the past 30 days. In the fully adjusted model, houselessness (AOR=2.27, 95%CI[1.48, 3.48]), a positive test result for Hepatitis C infection (AOR=1.73 95%CI[1.18, 2.52]) and heroin/fentanyl use (AOR= 8.58 95%CI [3.01, 24.50]) were associated with an increased risk of reporting past 30-day overdose, while having a high-school education or less was associated with reduced odds of overdose (AOR=0.52, 95% CI[0.37, 0.74])., Conclusion: As in urban areas, houselessness, Hepatitis C infection, and the use of heroin and fentanyl were significant correlates of overdose. Widespread access to overdose prevention interventions - including fentanyl test strips and naloxone - is critical in this rural context, with particular outreach needed to unhoused populations, people living with Hepatitis C, and people using opioids., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Introduction: Chronic pain and associated interference with daily activities are common in the military and impact Force readiness. Chronic pain affects one-third of service members and is a leading cause of medical non-readiness (MNR) in the military. Research suggests that underlying psychological mechanisms related to trait coping styles and pain interference (PI) affect functional outcomes, but little research exists examining this relationship within an Army population. The purpose of this study was to examine the combined effects of PI and coping on U.S. Army soldier readiness by using annual well-being data from the Global Assessment Tool (GAT) and medical non-readiness (MNR) based on duty restriction records., Methods: The sample comprised 866,379 soldiers who completed the GAT between 2014 and 2017 with no duty restrictions at the time of baseline GAT completion; subjects were observed through 2018 for duty restrictions. Parametric survival regression models with a Weibull distribution predicted demographic-adjusted hazards of MNR by dichotomized PI (no PI/PI) and beneficial/non-beneficial use of GAT coping components (good coping, bad coping, catastrophizing-flexibility, and catastrophizing-hopelessness). Incident MNR was evaluated for all duty restrictions, and stratified by selected body systems (upper extremity, lower extremity, psychiatric)., Results: Among soldiers with PI, hazards were higher in those reporting non-beneficial coping styles (bad coping, hopelessness) and lower in those reporting beneficial coping styles (good coping, flexibility). Across all coping styles, PI/coping interactions were particularly strong for catastrophizing-hopelessness and when examining MNR from psychiatric conditions., Discussion: These findings suggest some synergistic associations between pain and coping that may impact pain-related occupational disability. Coping skills may be an effective interventional target for chronic pain reduction/prevention within military programs, such as the Master Resilience Training Course offered to soldiers in the Army. Further research should assess whether early coping style interventions can reduce pain-related outcomes., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2023 Kegel, Kazman, Clifton, Emanuele, Nelson and Deuster.)

Introduction: Minimising temporary and permanent disability associated with musculoskeletal conditions (MSK-D) is critical to the mission of the US Army. Prior research has identified potentially actionable risk factors for overall military disability and its MSK-D subset, including elevated body mass index, tobacco use and physical fitness. However, prior work does not appear to have addressed the impact of these factors on MSK-D when controlling for a full range of factors that may affect health behaviours, including aptitude scores that may serve as a proxy for health literacy. Identifying risk factors for MSK-D when providing control for all such factors may inform efforts to improve military readiness., Methods: We studied 494 757 enlisted Army soldiers from 2014 to 2017 using a combined medical and administrative database. Leveraging data from the Army's digital 'eProfile' system of duty restriction records, we defined MSK-D as the first restriction associated with musculoskeletal conditions and resulting in the inability to deploy or train. We used multivariable Cox proportional hazards regression to assess the associations between incident MSK-D and selected risk factors including aptitude scores, physical fitness test scores, body mass index and tobacco use., Results: Among the subjects, 281 278 (45.14%) experienced MSK-D. In the MSK-D hazards model, the highest effect size was for failing the physical fitness test (adjusted HR=1.63, 95% CI 1.58 to 1.67, p<0.001) compared with scoring ≥290 points., Conclusions: The analysis revealed the strongest associations between physical fitness and MSK-D. Additional efforts are warranted to determine potential mechanisms for the observed associations between selected factors and MSK-D., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. No commercial re-use. See rights and permissions. Published by BMJ.)

Context: The US Army embedded injury-prevention experts (IPEs), specifically athletic trainers and strength and conditioning coaches, into initial entry training (IET) to limit musculoskeletal (MSK) conditions and their negative consequences. However, little is known about the financial impact of IPEs., Objective: To assess whether IPEs were associated with fewer sunk training costs due to MSK-related early discharges from service., Design: Retrospective cohort study., Setting: Database of US Army soldiers' administrative, medical, and readiness records., Patients or Other Participants: A total of 198 166 soldiers (age = 20.7 ± 3.2 years, body mass index = 24.4 ± 3.5 kg/m2) who began IET during 2014 to 2017., Main Outcome Measure(s): Early discharge from service was defined as occurring within 6 months of beginning IET. All IET sites employed IPEs from 2011 to 2017, except for 2 sites during April to November 2015. Soldiers who began IET at these 2 sites during these times were categorized as not having IPE exposure. All others were categorized as having IPE exposure. The unadjusted association between IPE access and MSK-related early discharge from service was assessed using logistic regression. Financial impact was assessed by quantifying differences in yearly sunk costs between groups with and those without IPE exposure and subtracting IPE hiring costs., Results: Among 14 094 soldiers without IPE exposure, 2.77% were discharged early for MSK-related reasons. Among 184 072 soldiers with IPE exposure, 1.01% were discharged. Exposure to IPEs was associated with reduced odds of MSK-related early discharge (odds ratio = 0.36, 95% CI = 0.32, 0.40, P < .001) and a decrease in yearly sunk training costs of $11.19 to $20.00 million., Conclusions: Employing IPEs was associated with reduced sunk costs because of fewer soldiers being discharged from service early for MSK-related reasons. Evidence-based recommendations should be developed for guiding policy on the roles and responsibilities of IPEs in the military to reduce negative outcomes from MSK conditions and generate a positive return on investment., (© by the National Athletic Trainers’ Association, Inc.)

Tri-plane corrective Lapidus surgery has been described as advantageous with respect to its anatomic basis and outcomes. Because the procedure has been broadly publicized, changes in overall Lapidus procedure rates due to increased numbers of patients opting for the tri-plane approach could have occurred. Data supporting this possibility appears lacking. We employed official personnel and health records of the total active-duty US military to conduct a retrospective cohort study of Lapidus surgery rates before and after the advent of the tri-plane corrective Lapidus procedure. Least-squares and locally-weighted scatterplot smoother regression functions were used to confirm time trends. Sociodemographic and occupational traits of Lapidus patients were compared using 2-sided t tests and chi square tests. Lapidus surgery rates among hallux valgus patients decreased during 2014 to 2016 and increased during 2017 to 2021. While multiple factors might explain these trends, they coincide with the advent of and advocacy for tri-plane Lapidus surgery. The results support the possibility that its rise influenced overall Lapidus rates in this population. As these findings represent limited evidence of such an influence, further research is required to confirm a causal link. If such a link is found, and if the ongoing research suggests that superior outcomes are associated with tri-plane Lapidus surgery, substantial implications could exist for this population. Benefits might include enhanced medical readiness due to the importance of lower extremity function during military duties. Additional research is needed to confirm the impact of the procedure and to determine whether Lapidus surgery rate patterns in civilian populations mirror these findings., (Published by Elsevier Inc.)

Purpose: The advantages of minimally invasive inguinal hernia repair (MIHR) over open hernia repair (OHR) continue to be debated. We compared MIHR to OHR by utilizing the Army Physical Fitness Test (APFT) as an outcome measure., Methods: The APFT is a three-component test scored on a normalized 300 point scale taken semiannually by active-duty military. We identified 1119 patients who met inclusion criteria: 588 in the OHR group and 531 in the MIHR group. Changes in APFT scores, time on post-operative duty restrictions (military profile), and time interval to first post-operative APFT were compared using regression analysis., Results: Postoperatively, no APFT score change difference was observed between the OHR or MIHR groups (- 7.3 ± 30 versus - 5.5 ± 27.7, p = 0.2989). Service members undergoing OHR and MIHR underwent their first post-operative APFT at equal mean timeframes (6.6 ± 5 months versus 6.7 ± 5.1, p = 0.74). No difference was observed for time in months spent on an official temporary duty restriction (military profile) for either OHR or MIHR (0.16 ± 0.16 versus 0.15 ± 0.17, p = 0.311). On adjusted regression analysis, higher pre-operative APFT scores and BMI ≥ 30 were independently associated with reduction in post-operative APFT scores. Higher-baseline APFT scores were independently associated with less time on a post-operative profile, whereas higher BMI (≥ 30) and lower rank were independently associated with longer post-operative profile duration. Higher-baseline APFT scores and lower rank were independently associated with shorter time intervals to the first post-operative APFT., Conclusion: Overall, no differences in post-operative APFT scores, military profile time, or time to first post-operative APFT were observed between minimally invasive or open hernioplasty in this military population., (© 2022. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

Objectives. To describe health-related behaviors or indicators associated with overall health and well-being using the Global Assessment Tool (GAT), a health behavior and psychosocial questionnaire completed annually by US Army personnel. Methods. We analyzed GAT responses from 2014 to 2018, consisting of 367 000 to 449 000 respondents per year. We used generalized estimating equations to predict the presence of each health behavior or indicator, aggregated by year and stratified on various demographics. Results. Key findings included decreases from 2014 to 2018 in risky health behaviors such as hazardous drinking (7.5% decrease) and tobacco use (7.9% decrease), dietary supplement use (5.0% to 10.6% decrease, depending on type), self-reported musculoskeletal injury (5.1% decrease), and pain interference (3.6% decrease). Physical activity, sleep, and nutritional habits largely remained consistent over time. Conclusions. In the Army, tobacco, alcohol, and risky dietary supplement usage appears to be declining, whereas lifestyle health behaviors have been stable. Whether these trends reflect responses to health education is unknown. The GAT provides useful insights into the health of the Army, which can be leveraged when developing health-related educational programs and policies. Public Health Implications. Health behaviors that have changed less over time (e.g., nutrition, sleep) may require novel approaches compared with those that changed more (e.g., dietary supplement use, drinking). ( Am J Public Health . 2021;111(11):2064-2074. https://doi.org/10.2105/AJPH.2021.306456).

Objective: To compare the epidemiology of concussion between athletes who are deaf or hard-of-hearing (D/HoH) and athletes who are hearing., Design: Descriptive epidemiology study., Setting: Data were collected from 2 Division III athletic programs. One institution is the world's only university designed to be barrier-free for students who are D/HoH., Participants: Six hundred ninety-three athletes who are D/HoH and 1284 athletes who are hearing were included in this study. Athletes participated in collegiate athletics during the 2012 to 2013 through the 2016 to 2017 academic years., Interventions: Concussion data were provided by the athletic training staff at each institution., Main Outcome Measures: Concussion counts, concussion rate, and injury rate ratios (IRRs) with 95% confidence intervals (95% CIs)., Results: Thirty athletes who are D/HoH and 104 athletes who are hearing suffered concussions. Athletes who are hearing had an increased injury rate compared with athletes who are D/HoH for all sports combined (IRR = 1.87, 95% CI, 1.26-2.78). Football athletes who are hearing also had an increased injury rate compared with football athletes who are D/HoH (IRR = 3.30, 95% CI, 1.71-6.37). Concussion rate was higher for male athletes who are hearing than male athletes who are D/HoH (IRR = 2.84, 95% CI, 1.62-4.97). No other significant differences regarding concussion risk were identified., Conclusions: Athletes who are D/HoH in sex-comparable sports may not have a higher rate of concussion than athletes who are hearing. Rate of concussion in football may be greater among athletes who are hearing compared with athletes who are D/HoH., Competing Interests: The authors report no conflicts of interest., (Copyright © 2018 Wolters Kluwer Health, Inc. All rights reserved.)

Introduction: Musculoskeletal injuries (MSK-I) in the U.S. military accounted for more than four million medical encounters in 2017. The Military Entrance Processing Screen to Assess Risk of Training (MEPSTART) was created to identify MSK-I risk during the first 180 days of military service., Methods: Active duty applicants to the United States Army, Navy, Air Force, and Marine Corps between February 2013 and December 2014 who consented completed a behavioral and injury history questionnaire and the MEPSTART screen [Functional Movement Screen (FMS), Y-Balance Test (YBT), Landing Error Scoring System (LESS), and Overhead Squat assessment (OHS)] the day they shipped to basic training. Male (n = 1,433) and Female (n = 281) applicants were enrolled and MSK-I were tracked for 180 days. Binomial logistic regression and multivariate Cox proportional hazards modeling were used to assess relationships among MEPSTART screens and MSK-I independent of age, BMI, sex, Service, injury history, and smoking status. Analyses were finalized and performed in 2017., Results: The only functional screen related to injury was the LESS score. Compared to those with good LESS scores, applicants with poor LESS scores had lower odds of MSK-I (OR = 0.54, 95% CI = 0.30-0.97, p = 0.04), and a lower instantaneous risk of MSK-I during the first 180 d (HR = 0.58, 95%CI = 0.34-0.96, p = 0.04). However, secondary receiver operator characteristic (ROC) analyses revealed poor discriminative value (AUC = 0.49, 95%CI = 0.43-0.54)., Conclusions: Functional performance did not predict future injury risk during the first 180 days of service. Poor LESS scores were associated with lower injury risk, but ROC analyses revealed little predictive value and limited clinical usefulness. Comprehensive risk reduction strategies may be preferable for mitigating MSK-I in military training populations., (Published by Oxford University Press on behalf of the Association of Military Surgeons of the United States 2019. This work is written by (a) US Government employee(s) and is in the public domain in the US.)

Background: Upper extremity injuries commonly occur in baseball players, and can often necessitate surgical interventions. Athletes recovering from previous surgeries may be at greater risk of a secondary injury due to potential residual deficits in global movement. Identifying individuals with residual movement dysfunction following surgery during a pre-participation examination may help health care professionals identify baseball players who may be at a greater risk of re-injury in their throwing arms so that appropriate interventions can be developed., Purpose: The purpose of this study was to assess relationships between history of shoulder or elbow surgeries and Functional Movement Screen™ (FMS™) shoulder mobility scores or Selective Functional Movement Assessment (SFMA) upper extremity patterns in collegiate baseball players., Study Design: Cohort study., Methods: One hundred seventy-six healthy, male, Division III collegiate baseball players (mean age = 19.65 ± 1.52 years) underwent preseason screening using the FMS™ shoulder mobility screen, and SFMA upper extremity patterns. Total FMS™ scores were dichotomized into "good" and "poor" groups (good = 2 or 3, poor = 0 or 1). SFMA scores were dichotomized into "good" and "poor" groups (good = functional non-painful (FN), poor = dysfunctional painful (DP), dysfunctional non-painful (DN), and functional painful (FP). Dichotomized FMS™ and SFMA scores were compared to questionnaire data regarding history of shoulder or elbow surgeries., Results: Thirty participants (17%) reported a previous shoulder or elbow surgery in their dominant arms. Past surgeries in the shoulder or elbow were not related to FMS™ (odds ratio [OR] = 0.74, 95% confidence interval [CI] = 0.30, 1.82), p = 0.52) or SFMA performance (OR = 0.93, 95%CI = 0.38, 2.27, p = 0.88) independent of grade and playing position., Conclusion: History of shoulder or elbow surgery was not related to performance on the FMS™ shoulder mobility test or SFMA upper extremity patterns. Differences in the dates of surgery at the time of testing, and sport-specific adaptations of the upper extremities that are common in baseball players due to the cumulative tissue stress from years of throwing at the collegiate level, may explain these insignificant findings., Level of Evidence: Level 3.

Context: The advent of Web-based sports injury surveillance via programs such as the High School Reporting Information Online system and the National Collegiate Athletic Association Injury Surveillance Program has aided the acquisition of boys' and men's basketball injury data., Objective: To describe the epidemiology of injuries sustained in high school boys' basketball in the 2005-2006 through 2013-2014 academic years and collegiate men's basketball in the 2004-2005 through 2013-2014 academic years using Web-based sports injury surveillance., Design: Descriptive epidemiology study., Setting: Online injury surveillance from basketball teams of high school boys (annual average = 100) and collegiate men (annual average = 55)., Patients or Other Participants: Boys' and men's basketball players who participated in practices and competitions during the 2005-2006 through 2013-2014 academic years in high school or the 2004-2005 through 2013-2014 academic years in college., Main Outcome Measures: Athletic trainers collected time-loss (≥24 hours) injury and exposure data. Injury rates per 1000 athlete-exposures (AEs) were calculated. Injury rate ratios (IRRs) with 95% confidence intervals (CIs) compared injury rates by school size or division, time in season, event type, and competition level., Results: The High School Reporting Information Online system documented 3056 time-loss injuries during 1 977 480 AEs; the National Collegiate Athletic Association Injury Surveillance Program documented 4607 time-loss injuries during 868 631 AEs. The injury rate was higher for college than for high school (5.30 versus 1.55/1000 AE; IRR = 3.43; 95% CI = 3.28, 3.59). The injury rate was higher for competitions than for practices in both high school (IRR = 2.38; 95% CI = 2.22, 2.56) and college (IRR = 2.02; 95% CI = 1.90, 2.14). The most common injuries at both levels were ligament sprains, muscle/tendon strains, and concussions; most injuries affected the ankle, knee, and head/face. Injuries were most often caused by contact with another player or noncontact mechanisms., Conclusions: Injury rates were greater among collegiate players compared with high school players and were greater during competitions than practices at both levels. Distributions of injuries by body part, diagnoses, and mechanisms of injury were similar, suggesting that athletes at both levels may benefit from similar injury-prevention strategies.

Context: The advent of Web-based sports injury surveillance via programs such as the High School Reporting Information Online system and the National Collegiate Athletic Association Injury Surveillance Program has aided the acquisition of girls' and women's basketball injury data., Objective: To describe the epidemiology of injuries sustained in high school girls' basketball in the 2005-2006 through 2013-2014 academic years and collegiate women's basketball in the 2004-2005 through 2013-2014 academic years using Web-based sports injury surveillance., Design: Descriptive epidemiology study., Setting: Online injury surveillance from basketball teams in high school girls (annual average = 100) and collegiate women (annual average = 57)., Patients or Other Participants: Girls' and women's basketball players who participated in practices and competitions during the 2005-2006 through 2013-2014 academic years in high school or the 2004-2005 through 2013-2014 academic years in college., Main Outcome Measure(s): Certified athletic trainers collected time-loss (≥24 hours) injury and exposure data. Injury rates per 1000 athlete-exposures (AEs) were calculated. Injury rate ratios (IRRs) with 95% confidence intervals (CIs) were used to compare injury rates by school size or division, time in season, event type, and competition level., Results: The High School Reporting Information Online system documented 2930 time-loss injuries during 1 609 733 AEs; the National Collegiate Athletic Association Injury Surveillance Program documented 3887 time-loss injuries during 783 600 AEs. The injury rate was higher in college than in high school (4.96 versus 1.82/1000 AEs; IRR = 2.73; 95% CI = 2.60, 2.86). The injury rate was higher in competitions than in practices for both high school (IRR = 3.03; 95% CI = 2.82, 3.26) and collegiate (IRR = 1.99; 95% CI = 1.86, 2.12) players. The most common injuries at both levels were ligament sprains, concussions, and muscle/tendon strains; the majority of injuries affected the ankle, knee, and head/face. These injuries were often caused by contact with another player or a noncontact mechanism., Conclusions: Injury rates were higher in collegiate than in high school athletes and in competitions than in practices. Similarities in distributions of injuries by body parts, specific diagnoses, and mechanisms of injury suggest that both levels may benefit from similar injury-prevention strategies.

Context:: Data regarding the epidemiology of emergency-transport incidents (ETIs) of patients with sport-related injuries are lacking. Understanding the use of emergency services by athletic trainers can help improve emergency preparedness and prehospital care for injured student-athletes., Objective:: To determine the frequencies and types of ETIs resulting from athletic participation., Design:: Descriptive epidemiology study., Setting:: Participating colleges and high schools during 2009-2010 to 2014-2015 and 2011-2012 to 2013-2014, respectively., Patients or Other Participants:: Student-athletes in 23 high school and 25 intercollegiate sports., Main Outcome Measure(s):: Data on injuries requiring emergency transport were collected by each team's athletic trainer via their respective online injury-tracking software. Athletic trainers also collected data on athlete-exposures (AEs). Emergency-transport incident frequencies and injury rates per 10 000 AEs with 95% confidence intervals (CIs) were reported. For each ETI, the sport, body part, injury mechanism, and final diagnosis were recorded., Results:: A total of 339 and 146 ETIs were reported in collegiate and high school players, respectively. Collegiate women's ice hockey had the highest ETI rate (1.28/10 000 AEs; 95% CI = 0.71, 1.86). In high school, football had the highest rate at 0.80 per 10 000 AEs (95% CI = 0.64, 0.97). Athletes with head or face injuries required the most transports in college (n = 71, 20.9%) and high school (n = 33, 22.6%) across all sports. Strains (n = 50, 14.7%) and fractures (n = 35, 24.0%) were the leading diagnoses for patients undergoing transport in college and high school, respectively., Conclusions:: Athletic trainers should maintain a high level of emergency preparedness when working with sports that have high rates and numbers of ETIs. Athletes with injuries to the head/face required the most frequent transport across competition levels. Athletic trainers should have the appropriate equipment and protocols in place to handle these patients. Future researchers should examine the differences between field and hospital diagnoses to help improve prehospital care and decrease the likelihood of unnecessary emergency transports.

Context:   The fourth edition of the Preparticipation Physical Evaluation recommends functional testing for the musculoskeletal portion of the examination; however, normative data across sex and grade level are limited. Establishing normative data can provide clinicians reference points with which to compare their patients, potentially aiding in the development of future injury-risk assessments and injury-mitigation programs., Objective:   To establish normative functional performance and limb-symmetry data for high school-aged male and female athletes in the United States., Design:   Cross-sectional study., Setting:   Athletic training facilities and gymnasiums across the United States., Patients or Other Participants:   A total of 3951 male and female athletes who participated on high school-sponsored basketball, football, lacrosse, or soccer teams enrolled in this nationwide study., Main Outcome Measure(s):   Functional performance testing consisted of 3 evaluations. Ankle-joint range of motion, balance, and lower extremity muscular power and landing control were assessed via the weight-bearing ankle-dorsiflexion-lunge, single-legged anterior-reach, and anterior single-legged hop-for-distance (SLHOP) tests, respectively. We used 2-way analyses of variance and χ 2 analyses to examine the effects of sex and grade level on ankle-dorsiflexion-lunge, single-legged anterior-reach, and SLHOP test performance and symmetry., Results:   The SLHOP performance differed between sexes (males = 187.8% ± 33.1% of limb length, females = 157.5% ± 27.8% of limb length; t = 30.3, P < .001). A Cohen d value of 0.97 indicated a large effect of sex on SLHOP performance. We observed differences for SLHOP and ankle-dorsiflexion-lunge performance among grade levels, but these differences were not clinically meaningful., Conclusions:   We demonstrated differences in normative data for lower extremity functional performance during preparticipation physical evaluations across sex and grade levels. The results of this study will allow clinicians to compare sex- and grade-specific functional performances and implement approaches for preventing musculoskeletal injuries in high school-aged athletes.

Background: The shoulder mobility screen of the Functional Movement Screen™ (FMS™) and the upper extremity patterns of the Selective Functional Movement Assessment (SFMA) assess global, multi-joint movement capabilities in the upper-extremities. Identifying which assessment can most accurately determine if baseball players are at an increased risk of experiencing overuse symptoms in the shoulder or elbow throughout a competitive season may reduce throwing-related injuries requiring medical attention., Purpose: The purpose of this study was to determine if preseason FMS™ or SFMA scores were related to overuse severity scores in the shoulder or elbow during the preseason and competitive season., Study Design: Cohort study., Methods: Sixty healthy, male, Division III collegiate baseball players (mean age = 20.1 ± 2.0 years) underwent preseason testing using the FMS™ shoulder mobility screen, and SFMA upper extremity patterns. Their scores were dichotomized into good and bad movement scores, and were compared to weekly questionnaires registering overuse symptoms and pain severity in the shoulder or elbow during the season., Results: Poor FMS™ performance was associated with an increased likelihood of experiencing at least one overuse symptom during the preseason independent of grade and position (adjusted odds ratio [OR] = 5.14, p = 0.03). Poor SFMA performance was associated with an increased likelihood of experiencing at least one overuse symptom during the preseason (adjusted OR = 6.10, p = 0.03) and during the competitive season (adjusted OR = 17.07, p = 0.03) independent of grade and position., Conclusion: FMS™ shoulder mobility and SFMA upper extremity pattern performance were related to the likelihood of experiencing overuse symptoms during a baseball season. Participants with poor FMSTM performances may be more likely to experience at least one overuse symptom in their shoulder or elbow during the preseason. Additionally, individuals with poor SFMA performances may be more likely to report overuse symptoms during the preseason or competitive season., Level of Evidence: Level 3.

Context:   Variations in knee-sprain incidence among competition levels are unclear but may help inform prevention strategies in American football players., Objective:   To describe the epidemiology of knee sprains in youth, high school, and collegiate football players., Design:   Descriptive epidemiology study., Setting:   Injury and athlete-exposure (AE) data were collected from 3 injury-surveillance programs at the youth, high school, and collegiate competition levels., Patients or Other Participants:   Data from 310 youth, 184 high school, and 71 collegiate football team-seasons were collected during the 2012 through 2014 seasons., Main Outcome Measure(s):   Knee-sprain rates and risks were calculated for each competition level. Injury rate ratios (IRRs) and risk ratios (RRs) compared knee-sprain rates by competition level. Injury proportion ratios (IPRs) compared differences in surgery needs, recurrence, injury mechanism, and injury activity by competition level., Results:   Knee-sprain rates in youth, high school, and collegiate football were 0.16/1000 AEs, 0.25/1000 AEs, and 0.69/1000 AEs, respectively. Knee-sprain rates increased as the competition level increased (high school versus youth: IRR = 1.60; 95% confidence interval [CI] = 1.12, 2.30; collegiate versus high school: IRR = 2.73; 95% CI = 2.38, 3.96). Knee-sprain risk was highest in collegiate (4.3%), followed by high school (2.0%) and youth (0.5%) athletes. Knee-sprain risk increased as the competition level increased (high school versus youth: RR = 3.73; 95% CI = 2.60, 5.34; collegiate versus high school: RR = 2.14; 95% CI = 1.83, 2.51). Collegiate football had the lowest proportion of knee sprains that were noncontact injuries (collegiate versus youth: IPR = 0.54; 95% CI = 0.31, 0.95; collegiate versus high school: IPR = 0.59; 95% CI = 0.44, 0.79) and the lowest proportion that occurred while being tackled (collegiate versus youth: IPR = 0.44; 95% CI = 0.26, 0.76; collegiate versus high school: IPR = 0.71; 95% CI = 0.51, 0.98)., Conclusions:   Knee-sprain incidence was highest in collegiate football. However, level-specific variations in the distributions of knee sprains by injury activity may highlight the need to develop level-specific policies and prevention strategies that ensure safe sports play.

Background: Variations in ankle injury rates and distributions among competition levels are unclear, but such data may help inform strategies to prevent ankle sprains during American football., Purpose: To describe the epidemiological patterns of ankle sprains in youth, high school (HS), and collegiate American football., Study Design: Descriptive epidemiological study., Methods: Data regarding youth, HS, and college football athletes were collected from 3 injury surveillance programs: (1) the Youth Football Safety Study (YFSS), (2) the National Athletic Treatment, Injury and Outcomes Network (NATION), and (3) the National Collegiate Athletic Association (NCAA) Injury Surveillance Program (ISP). During the 2012-2014 seasons, the YFSS, NATION, and NCAA ISP included 310, 184, and 71 football team-seasons, respectively. Athletic trainers (ATs) attended each practice and game and reported injuries and athlete-exposures (AEs) via their preferred injury documentation application. Ankle sprain rates for each type of ankle sprain were calculated overall, by event type (ie, practices and games), and specifically for severe injuries (ie, participation restriction time >21 days) and recurrent injuries (as defined by ATs). Rate ratios (RRs) were used to compare ankle sprain rates by competition level and event type. Injury proportion ratios (IPRs) were used to compare differences in severity, surgical needs, recurrence, injury mechanism, and injury activity by competition level. RRs and IPRs with 95% confidence intervals excluding 1.00 were considered statistically significant., Results: A total of 124, 897, and 643 ankle sprains were reported in youth, HS, and college football, respectively. This led to respective rates of 0.59, 0.73, and 1.19 sprains per 1000 AEs. The ankle sprain rate in college football was higher than the rates in HS (RR = 1.64; 95% CI, 1.48-1.82) and youth (RR = 2.01; 95% CI, 1.65-2.43) football. The proportion of ankle sprains that were recurrent in youth football was higher than the proportions in HS (IPR = 2.73; 95% CI, 1.68-4.50) and college (IPR = 2.19; 95% CI, 1.33-3.61) football., Conclusion: Ankle sprain rates were highest in college athletes. However, level-specific variations in ankle sprain severity and recurrence may highlight the need to develop level-specific policies and prevention strategies to reduce injury incidence.

Objective: A stated goal of the preparticipation physical evaluation (PPE) is to reduce musculoskeletal injury, yet the musculoskeletal portion of the PPE is reportedly of questionable use in assessing lower extremity injury risk in high school-aged athletes. The objectives of this study are: (1) identify clinical assessment tools demonstrated to effectively determine lower extremity injury risk in a prospective setting, and (2) critically assess the methodological quality of prospective lower extremity risk assessment studies that use these tools., Data Sources: A systematic search was performed in PubMed, CINAHL, UptoDate, Google Scholar, Cochrane Reviews, and SportDiscus. Inclusion criteria were prospective injury risk assessment studies involving athletes primarily ages 13 to 19 that used screening methods that did not require highly specialized equipment. Methodological quality was evaluated with a modified physiotherapy evidence database (PEDro) scale., Main Results: Nine studies were included. The mean modified PEDro score was 6.0/10 (SD, 1.5). Multidirectional balance (odds ratio [OR], 3.0; CI, 1.5-6.1; P < 0.05) and physical maturation status (P < 0.05) were predictive of overall injury risk, knee hyperextension was predictive of anterior cruciate ligament injury (OR, 5.0; CI, 1.2-18.4; P < 0.05), hip external:internal rotator strength ratio of patellofemoral pain syndrome (P = 0.02), and foot posture index of ankle sprain (r = -0.339, P = 0.008)., Conclusions: Minimal prospective evidence supports or refutes the use of the functional musculoskeletal exam portion of the current PPE to assess lower extremity injury risk in high school athletes. Limited evidence does support inclusion of multidirectional balance assessment and physical maturation status in a musculoskeletal exam as both are generalizable risk factors for lower extremity injury., Competing Interests: The authors report no conflicts of interest.

Context: Musculoskeletal injury-prediction methods vary and may have limitations that affect the accuracy of results and clinical meaningfulness., Background: Research examining injury risk factors is meaningful, but attempting to extrapolate injury risk from studies that do not prospectively assess injury occurrence may limit clinical applications. Injury incidence is a vital outcome measure, which allows for the appropriate interpretation of injury-prediction analyses; a lack of injury-incidence data may decrease the accuracy and increase the uncertainty of injury-risk estimates. Extrapolating results that predict an injury risk factor to predicting actual injuries may lead to inappropriate clinical decision-making models., Conclusions: Improved understanding of the limitations of injury-prediction methods, specifically those that do not prospectively assess injuries, will allow clinicians to better assess the clinical meaningfulness of the results.

Background: The Functional Movement Screen (FMS™) has been suggested for use in predicting injury risk in active populations, but time constraints may limit use of the screening test battery. Identifying one component of the FMS™ that can predict which individuals may perform poorly on the entire test, and therefore should undergo the full group of screening maneuvers, may reduce time constraints and increase pre-participation screening utilization., Purpose: The purpose of this study was to determine if performance on the FMS™ overhead deep squat test (DS) could predict performance on the entire FMS™., Study Design: Cohort study., Methods: One hundred and three collegiate athletes underwent offseason FMS™ testing. The DS and adjusted FMS™ composite scores were dichotomized into low performance and high performance groups with athletes scoring below 2 on the DS categorized as low performance, and athletes with adjusted FMS™ composite scores below 12 categorized as low performance. Scores of 2 or above and 12 or above were considered high performances for the DS test and adjusted FMS™ composite score respectively, and therefore low risk for movement dysfunction and potentially, injury., Results: Individuals categorized as low performance as a result of the DS test had lower adjusted FMS™ composite scores (p < 0.001). DS scores were positively correlated with adjusted FMS™ composite scores (ρ = 0.50, p < 0.001). Binomial logistic regression identified an odds ratio of 3.56 (95% CI: 1.24, 10.23, p = 0.018) between DS and FMS™ performance categories., Conclusions: Performance on the DS test may predict performance on the FMS™ and help identify individuals who require further musculoskeletal assessment. Further research is needed to determine if DS performance can predict asymmetries during the FMS™., Level of Evidence: Level 3.

Staphylococcus aureus community-acquired pneumonia is often associated with influenza or an influenza-like syndrome. Morbidity and mortality due to methicillin-resistant S. aureus (MRSA) or influenza and pneumonia, which includes bacterial co-infection, are among the top causes of death by infectious diseases in the United States. We developed a non-lethal influenza A virus (IAV) (H3N2)/S. aureus co-infection model in cynomolgus macaques (Macaca fascicularis) to test the hypothesis that seasonal IAV infection predisposes non-human primates to severe S. aureus pneumonia. Infection and disease progression were monitored by clinical assessment of animal health; analysis of blood chemistry, nasal swabs, and X-rays; and gross pathology and histopathology of lungs from infected animals. Seasonal IAV infection in healthy cynomolgus macaques caused mild pneumonia, but unexpectedly, did not predispose these animals to subsequent severe infection with the community-associated MRSA clone USA300. We conclude that in our co-infection model, seasonal IAV infection alone is not sufficient to promote severe S. aureus pneumonia in otherwise healthy non-human primates. The implication of these findings is that comorbidity factors in addition to IAV infection are required to predispose individuals to secondary S. aureus pneumonia.

The impact of the Borrelia burgdorferi surface-localized immunogenic lipoprotein BBA66 on vector and host infection was evaluated by inactivating the encoding gene, bba66, and characterizing the mutant phenotype throughout the natural mouse-tick-mouse cycle. The BBA66-deficient mutant isolate, Bb(ΔA66), remained infectious in mice by needle inoculation of cultured organisms, but differences in spirochete burden and pathology in the tibiotarsal joint were observed relative to the parental wild-type (WT) strain. Ixodes scapularis larvae successfully acquired Bb(ΔA66) following feeding on infected mice, and the organisms persisted in these ticks through the molt to nymphs. A series of tick transmission experiments (n = 7) demonstrated that the ability of Bb(ΔA66)-infected nymphs to infect laboratory mice was significantly impaired compared to that of mice fed upon by WT-infected ticks. trans-complementation of Bb(ΔA66) with an intact copy of bba66 restored the WT infectious phenotype in mice via tick transmission. These results suggest a role for BBA66 in facilitating B. burgdorferi dissemination and transmission from the tick vector to the mammalian host as part of the disease process for Lyme borreliosis.

The Functional Movement Screen (FMS) is currently used for injury risk prediction, although researchers have not studied its relationships to injury risk factors. The purpose of this study was to compare FMS scores at rest to changes in static balance after exercise. Second, we examined FMS scores pre and post exercise. Twenty-five participants performed center of pressure (COP) measures and FMS testing. An acclimatization session for the FMS occurred on day 1, whereas day 2 involved COP measures for static balance and FMS testing before and after a 36-minute exercise protocol. Center of pressure standard deviations in the frontal (COPML-SD) and sagittal (COPAP-SD) planes, center of pressure velocity (COP-Velocity), center of pressure area (COP-Area), and FMS scores were recorded. No significant correlations occurred between preexercise FMS scores and change in COP measures. Preexercise hurdle step scores related to preexercise COPML-SD (p = -0.46), COPAP-SD (p = -0.43), and COP-Area (p = -0.50). Preexercise in-line lunge scores related to postexercise COPAP-SD (p = -0.44) and COP-Velocity (p = -0.39), whereas preexercise active straight leg raise (ASLR) scores related to postexercise COPML-SD (p = -0.46). Functional Movement Screen scores were not related to changes in static balance after exercise and may therefore not be useful to predict who will experience greater static balance deficits after exercise. Additionally, FMS scores did not differ before and after exercise. Clinicians aiming to identify injury risk from a general static balance standpoint may find the hurdle step, in-line lunge, and ASLR useful. Clinicians aiming to identify injury risk from a change in static balance standpoint may need to explore other screening tools.

The spirochetal agent of Lyme disease, Borrelia burgdorferi, is transmitted by bites of Ixodes ticks to mammalian reservoir hosts and humans. The mechanism(s) by which the organism is trafficked from vector to host is poorly understood. In this study, we demonstrate that a B. burgdorferi mutant strain deficient in the synthesis of the bba64 gene product was incapable of infecting mice via tick bite even though the mutant was (i) infectious in mice when introduced by needle inoculation, (ii) acquired by larval ticks feeding on infected mice, and (iii) able to persist through tick molting stages. This finding of a B. burgdorferi gene required for pathogen transfer and/or survival from the tick to the susceptible host represents an important breakthrough toward understanding transmission mechanisms involved for the Lyme disease agent.

Borrelia burgdorferi, the causative agent of Lyme disease in the United States, regulates numerous genes encoding lipoproteins on linear plasmid 54 in response to environmental cues. We analyzed a subset of these genes/proteins that were historically categorized as paralogous gene family 54 (BBA64, BBA65, BBA66, BBA68, BBA69, BBA70, BBA71, and BBA73) and found that the expression of several genes was influenced by the sigma(N)-sigma(S) regulatory cascade at the level of transcription and protein synthesis. Moreover, we established in this and a previous study that BBA65, BBA66, BBA69, BBA71, and BBA73 are temporally expressed during persistent infection of immunocompetent mice, as determined by quantitative real time-PCR of ear tissue, by enzyme-linked immunosorbent assay, and by immunoblotting. Correspondingly, BBA65, BBA66, BBA71, and BBA73 proteins were detectable in infectious B. burgdorferi B31 isolates but undetectable in noninfectious isolates. BBA65, BBA66, BBA71, and BBA73 proteins were also found to partition into the Triton X-114 detergent phase and were sensitive to protease treatment of intact cells, indicating that they are membrane associated and surface localized. Lastly, Southern blotting and PCR with specific gene primer/probes for BBA64, BBA65, BBA66, BBA71, and BBA73 suggest that many of these genes are conserved among the B. burgdorferi sensu lato isolates and the relapsing-fever Borrelia species. Together, the data presented suggest that these genes may play a part in Borrelia infection and/or pathogenicity that could extend beyond the sensu lato group.

Members of the Borrelia burgdorferi paralogous gene family 54 (pgf 54) are regulated by conditions simulating mammalian infection and are thought to be instrumental in borrelial host survival and pathogenesis. To explore the activities of these genes in vivo, a comprehensive analysis of pgf 54 genes BBA64, BBA65, and BBA66 was performed to assess the genetic stability, host antibody responses, and kinetics of gene expression in the murine model of persistent infection. DNA sequencing of pgf 54 genes obtained from re-isolates at 1 year postinfection demonstrated that all genes of this family are stable and do not undergo recombination to generate variant antigens during persistent infection. Antibodies against BBA64 and BBA66 appeared soon after infection and were detectable throughout the infection, suggesting that there was gene expression during infection. However, quantitative reverse transcription-PCR revealed that BBA64 gene expression was considerably decreased in Borrelia residing in the mouse ear tissue compared to the expression in cultured spirochetes by 20 days postinfection and that the levels of expression remained low throughout the infection. Conversely, transcription of the BBA65 and BBA66 genes was increased, and both of these genes were continuously expressed until 100 days postinfection; this was followed by periods of differential expression late in infection. The expression profile of the BBA64 gene suggests that this gene has an important role during tick-to-host transmission and early infection, whereas the expression profile of the BBA65 and BBA66 genes suggests that these genes have a role in persistent infection. The differential regulation of pgf 54 genes observed during infection may help confer a survival advantage during persistent infection, influencing mechanisms for B. burgdorferi dissemination, tissue tropism, or evasion of the adaptive immune response.

When Borrelia burgdorferi (Bb) is transmitted from a tick vector to a mammalian host the spirochaete alters gene expression, allowing for adaptation to the new host. We evaluated the regulation of paralogous gene family (pgf) 54 members in response to environmental cues and focused our efforts on determining the molecular mechanisms influencing bba66 expression. By qRT-PCR, bba65, bba66, bba71 and bba73 displayed regulation similar to ospC under mammalian-like conditions. Of the pgf 54 members, bba66 demonstrated the greatest and second greatest change in expression in response to pH or temperature shift respectively. Furthermore, Bb-infected mice and patients with early disseminated Lyme disease produced detectable antibodies to BBA66. A protein(s) active in Bb at pH 7 was able to interact with the bba66 upstream region and was specific as bba64 and ospC promoters were unable to out-compete for binding. bba66 promoter mapping revealed putative sigma70 and sigmaS consensus sequences, enabling us to narrow the protein binding site to a region within an imperfect inverted repeat upstream of the -35 region. Moreover, BBA66 production is associated with an infectious phenotype, and loss of either sigmaN or sigmaS resulted in loss of BBA66. Promoter-GFP fusion analysis indicated that the sigma70 and/or sigmaS consensus sequences alone were not sufficient to initiate transcription and a portion of the upstream inverted repeat was required. These results suggest a primary role for BBA66 in Bb transmission and infection.

We previously demonstrated that outer surface protein C (OspC) of Borrelia burgdorferi is essential for establishing mammalian infection. However, the role of OspC in mammalian infection is unknown. Here, we report experiments designed to distinguish between two models of OspC function in the mammalian host: (i) OspC fulfills an essential physiological role for growth and host adaptation or (ii) OspC provides a protective role for evasion of components of the innate immune response. We found that a B. burgdorferi ospC mutant, previously demonstrated to be noninfectious in both immunocompetent and SCID mice, could survive in the relatively immune-privileged environment of dialysis membrane chambers implanted within the peritoneum of a rat. The ospC mutant also adapts to the mammalian environment, as determined by the protein profiles of the chamber-cultivated spirochetes. Therefore, OspC does not appear to provide a physiological function for the survival of B. burgdorferi within the mammalian host. The second model, evasion of the innate immune system, was tested by assessing the infectivity of the ospC mutant in mice deficient for myeloid differentiation protein 88 (MyD88). Recent studies have shown that B. burgdorferi is prevented from reaching high cell numbers in the mammalian host by MyD88-dependent signaling pathways. The ospC mutant was incapable of infecting MyD88-deficient mice, suggesting that the role of OspC cannot be related solely to evasion of MyD88-mediated innate immunity. These results reiterate the importance of OspC in mammalian infection and eliminate simple models of function for this enigmatic protein.

Borrelia burgdorferi, the cause of Lyme disease, produces excessive amounts of membrane lipoproteins such as outer surface protein A (OspA) when grown in vitro, and consequently many low or moderately abundant proteins are underrepresented when cell lysates are examined by 2-DE. We analyzed the B. burgdorferi B31 proteome computationally and by IPG or modified NEPHGE after subcellular fractionation into membrane-associated and soluble proteins. The B. burgdorferi B31 theoretical proteome is comprised of 1623 proteins and has a mean pI of 8.36 and a median pI of 9.03 with 68% of the proteome possessing a pI >/=7.5. Separation of soluble proteins by IPG resulted in 205 individual spots and identification of 78 protein spots by MALDI-TOF MS. Separation by modified NEPHGE routinely resulted in approximately 185 soluble and 160 membrane protein spots with the identification of 88 individual protein spots combined by MALDI-TOF MS. Homologues to GroEL and aminopeptidase I were present in greater amounts in the membrane faction, with enolase at nearly equivalent amounts in the soluble and membrane fractions. Identification of proteins isolated and separated by such methods will enable future determination of proteome changes in membrane and soluble protein fractions as spirochetes adapt to their changing environments.

Infection of endothelial cells (EC) with Rickettsia rickettsii results in Rocky Mountain spotted fever, an acute illness characterized by systemic inflammation. Interleukin-8 (IL-8) and monocyte chemoattractant protein-1 (MCP-1) are important chemokines for activating neutrophils and monocytes, respectively, and recruiting these circulating immune cells to the sites of inflammation. In this study, we have measured the expression and secretion of these chemokines during R. rickettsii infection of cultured human EC. In comparison to uninfected controls, increased mRNA expression of IL-8 and MCP-1 in R. rickettsii-infected EC was evident as early as 3 h and was sustained up to 21 h. Subsequent analysis of culture supernatants revealed significantly enhanced secretion of both chemokines at 3, 8, and 18 h post-infection (5-28-fold increase in IL-8 and 4-16-fold increase in MCP-1). The presence of peptide-aldehyde compound MG132 to inhibit proteasome-mediated degradation of the inhibitory protein IkappaBalpha and synthetic peptide SN-50 to inhibit the nuclear translocation of nuclear factor-kappa B (NF-kappaB) resulted in significant inhibition of the chemokine response. Also, T24 cells expressing a super-repressor mutant of IkappaBalpha (to render NF-kappaB inactivatable) secreted significantly lower quantities of IL-8 than mock-transfected cells. A neutralizing antibody against IL-1alpha or an IL-1 specific receptor antagonist had no effect on the early phase of R. rickettsii-induced NF-kappaB activation and IL-8/ MCP-1 secretion at 3 h. Both of these treatments, however, diminished late-phase NF-kappaB activation by about 33% and only partially suppressed the infection-induced chemokine release at 21 h. Thus, while chemokine response early during the infection likely depends on the direct activation of NF-kappaB, subtle autocrine effects of newly synthesized IL-1alpha may contribute, in part, to the control of NF-kappaB activation and chemokine production at later times. These findings implicate a prominent role for host EC in recruiting immune cells to the site of inflammation during Rickettsia infection and provide important insights to further our understanding of the pathogenesis of spotted fever group rickettsioses.

Chlamydiae are obligate intracellular pathogens that efficiently induce their endocytosis by susceptible eukaryotic host cells. Recently, a Chlamydia trachomatis type III secreted effector protein, Tarp, was found to be translocated and tyrosine phosphorylated at the site of entry and associated with the recruitment of actin that coincides with endocytosis. C. trachomatis Tarp possesses up to six direct repeats of approximately 50 amino acids each. The majority of the tyrosine residues are found within this repeat region. Here we have ectopically expressed distinct domains of Tarp in HeLa 229 cells and demonstrated that tyrosine phosphorylation occurs primarily within the repeat region, while recruitment of actin is mediated by the C-terminal domain of the protein. A comparison of other sequenced chlamydial genomes revealed that each contains an ortholog of Tarp, although Chlamydia muridarum, Chlamydophila caviae, and Chlamydophila pneumoniae Tarp lack the large repeat region. Immunofluorescence and immunoblotting using an antiphosphotyrosine antibody show no evidence of phosphotyrosine at the site of entry of C. muridarum, C. caviae, and C. pneumoniae, although each species similarly recruits actin. Ectopic expression of full-length C. trachomatis and C. caviae Tarp confirmed that both recruit actin but only C. trachomatis Tarp is tyrosine phosphorylated. The data indicate that the C-terminal domain of Tarp is essential for actin recruitment and that tyrosine phosphorylation may not be an absolute requirement for actin recruitment. The results further suggest the potential for additional, unknown signal transduction pathways associated specifically with C. trachomatis.

Rocky Mountain spotted fever, a systemic tick-borne illness caused by the obligate intracellular bacterium Rickettsia rickettsii, is associated with widespread infection of the vascular endothelium. R. rickettsii infection induces a biphasic pattern of the nuclear factor-kappaB (NF-kappaB) activation in cultured human endothelial cells (ECs), characterized by an early transient phase at 3 h and a late sustained phase evident at 18 to 24 h. To elucidate the underlying mechanisms, we investigated the expression of NF-kappaB subunits, p65 and p50, and IkappaB proteins, IkappaBalpha and IkappaBbeta. The transcript and protein levels of p50, p65, and IkappaBbeta remained relatively unchanged during the course of infection, but Ser-32 phosphorylation of IkappaBalpha at 3 h was significantly increased over the basal level in uninfected cells concomitant with a significant increase in the expression of IkappaBalpha mRNA. The level of IkappaBalpha mRNA gradually returned toward baseline, whereas that of total IkappaBalpha protein remained lower than the corresponding controls. The activities of IKKalpha and IKKbeta, the catalytic subunits of IkappaB kinase (IKK) complex, as measured by in vitro kinase assays with immunoprecipitates from uninfected and R. rickettsii-infected ECs, revealed significant increases at 2 h after infection. The activation of IKK and early phase of NF-kappaB response were inhibited by heat treatment and completely abolished by formalin fixation of rickettsiae. The IKK inhibitors parthenolide and aspirin blocked the activities of infection-induced IKKalpha and IKKbeta, leading to attenuation of nuclear translocation of NF-kappaB. Also, increased activity of IKKalpha was evident later during the infection, coinciding with the late phase of NF-kappaB activation. Thus, activation of catalytic components of the IKK complex represents an important upstream signaling event in the pathway for R. rickettsii-induced NF-kappaB activation. Since NF-kappaB is a critical regulator of inflammatory genes and prevents host cell death during infection via antiapoptotic functions, selective inhibition of IKK may provide a potential target for enhanced clearance of rickettsiae and an effective strategy to reduce inflammatory damage to the host during rickettsial infections.

The obligate intracellular bacterium Chlamydia trachomatis rapidly induces its own entry into host cells. Initial attachment is mediated by electrostatic interactions to heparan sulfate moieties on the host cell, followed by irreversible binding to an unknown secondary receptor. This secondary binding leads to the recruitment of actin to the site of attachment, formation of an actin-rich, pedestal-like structure, and finally internalization of the bacteria. How chlamydiae induce this process is unknown. We have identified a high-molecular-mass tyrosine-phosphorylated protein that is rapidly phosphorylated on attachment to the host cell. Immunoelectron microscopy studies revealed that this tyrosine-phosphorylated protein is localized to the cytoplasmic face of the plasma membrane at the site of attachment of surface-associated chlamydiae. The phosphoprotein was isolated by immunoprecipitation with the antiphosphotyrosine antibody 4G10 and identified as the chlamydial protein CT456, a hypothetical protein with unknown function. The chlamydial protein (Tarp) appears to be translocated into the host cell by type III secretion because it is exported in a Yersinia heterologous expression assay. Phosphotyrosine signaling across the plasma membrane preceded the recruitment of actin to the site of chlamydial attachment and may represent the initial signal transduced from pathogen to the host cell. These results suggest that C. trachomatis internalization is mediated by a chlamydial type III-secreted effector protein.

A nonpathogenic mutant of Colletotrichum magna (path-1) was previously shown to protect watermelon (Citrullus lanatus) and cucumber (Cucumis sativus) seedlings from anthracnose disease elicited by wild-type C. magna. Disease protection was observed in stems of path-1-colonized cucurbits but not in cotyledons, indicating that path-1 conferred tissue-specific and/or localized protection. Plant biochemical indicators of a localized and systemic (peroxidase, phenylalanine ammonia-lyase, lignin, and salicylic acid) "plant-defense" response were investigated in anthracnose-resistant and -susceptible cultivars of cucurbit seedlings exposed to four treatments: (1) water (control), (2) path-1 conidia, (3) wild-type conidia, and (4) challenge conditions (inoculation into path-1 conidia for 48 h and then exposure to wild-type conidia). Collectively, these analyses indicated that disease protection in path-1-colonized plants was correlated with the ability of these plants to mount a defense response more rapidly and to equal or greater levels than plants exposed to wild-type C. magna alone. Watermelon plants colonized with path-1 were also protected against disease caused by Colletotrichum orbiculare and Fusarium oxysporum. A model based on the kinetics of plant-defense activation is presented to explain the mechanism of path-1-conferred disease protection.

The possibility that bacteria may have evolved strategies to overcome host cell apoptosis was explored by using Rickettsia rickettsii, an obligate intracellular Gram-negative bacteria that is the etiologic agent of Rocky Mountain spotted fever. The vascular endothelial cell, the primary target cell during in vivo infection, exhibits no evidence of apoptosis during natural infection and is maintained for a sufficient time to allow replication and cell-to-cell spread prior to eventual death due to necrotic damage. Prior work in our laboratory demonstrated that R. rickettsii infection activates the transcription factor NF-kappa B and alters expression of several genes under its control. However, when R. rickettsii-induced activation of NF-kappa B was inhibited, apoptosis of infected but not uninfected endothelial cells rapidly ensued. In addition, human embryonic fibroblasts stably transfected with a superrepressor mutant inhibitory subunit Ikappa B that rendered NF-kappa B inactivatable also underwent apoptosis when infected, whereas infected wild-type human embryonic fibroblasts survived. R. rickettsii, therefore, appeared to inhibit host cell apoptosis via a mechanism dependent on NF-kappa B activation. Apoptotic nuclear changes correlated with presence of intracellular organisms and thus this previously unrecognized proapoptotic signal, masked by concomitant NF-kappa B activation, likely required intracellular infection. Our studies demonstrate that a bacterial organism can exert an antiapoptotic effect, thus modulating the host cell's apoptotic response to its own advantage by potentially allowing the host cell to remain as a site of infection.