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Background: While tibia vara is a disorder of the proximal tibial physis, femoral deformity frequently contributes to the overall limb malalignment. Our purpose was to determine how femoral varus deformity in tibia vara responds to growth modulation, with/without lateral tension band plating (LTBP) to the femur., Methods: One-hundred twenty-seven limbs undergoing LTBP for tibia vara were reviewed. All had tibial LTBP and 35 limbs also had femoral LTBP for varus. Radiographs were measured for correction of the mechanical lateral distal femoral angle (mLDFA) and mechanical axis deviation (MAD). Preoperative-femoral varus was defined with an age-adjusted guide: mLDFA >95 degrees for 2 to below 4 years and mLDFA >90 degrees for 4 to 18 years. The 35 limbs having femoral LTBP were compared with 50 limbs with femoral varus and no femoral LTBP. In addition, 42 limbs that did not have preoperative-femoral varus were followed. Patients with early-onset (below 7 y) tibia vara were compared with those with late-onset (≥8 y). Outcome success was based on published age-adjusted mLDFA and MAD norms., Results: Following femoral LTBP, the mean mLDFA decreased from 98.0 to 87.1 degrees. All femurs had some improvement, with 28/35 femurs (80%) achieving complete correction. One limb, with late follow-up, overcorrected, requiring reverse (medial) femoral tension band plating.For the 50 limbs with femoral varus and only tibial LTBP, 16/22 limbs (73%) with early-onset and 11/28 limbs (39%) with late-onset completely corrected their femoral deformities. If the limb had preoperative-femoral varus, femoral LTBP statistically correlated with successful mLDFA correction and improvement of MAD, only in the late-onset group.Forty-two limbs, without preoperative-femoral varus, had no change in their mean mLDFA of 87 degrees. However, 4 femurs (10%) ended with posttreatment varus., Conclusions: Femoral LTBP is effective in correcting femoral varus deformity in the tibia vara. For femoral varus associated with late-onset tibia vara, femoral LTBP should be considered. Those that had femoral LTBP had statistically more successful femoral and overall limb varus correction. However, in early-onset tibia vara, with associated femoral varus, observation is warranted because 73% of femurs are corrected without femoral intervention. This study was underpowered to show additional improvement with femoral LTBP in the early-onset group. Even limbs with normal femoral alignment, should be observed closely for the development of femoral varus, during tibial LTBP treatment for tibia vara., Level of Evidence: Level III., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Angular deformity correction with tension band plating has not been as successful in early-onset tibia vara (EOTV) as it has been in other conditions. Our hypothesis is that perioperative factors can predict the success of lateral tibial tension band plating (LTTBP) in patients with EOTV., Methods: A retrospective review was performed at 7 centers evaluating radiographic outcomes of LTTBP in patients with EOTV (onset <7 y of age). Single-event tibial LTTBP outcome was assessed through medial proximal tibial angle (MPTA). The final limb alignment following comprehensive limb growth modulation (CLGM), which could include multiple procedures, was assessed by mechanical axis zone (MAZone), mechanical tibio-femoral angle (mTFA), and mechanical axis deviation (MAD). Preoperative age, weight, deformity severity, medial physeal slope, and Langenskiöld classification +/- modification were investigated as predictors of outcome. Success was defined as the correction or overcorrection to normal age-adjusted alignment. The minimum follow-up was 2 years except when deformity correction, skeletal maturity, or additional surgery occurred., Results: Fifty-two patients with 80 limbs underwent 115 tibial LTTBP procedures at a mean age of 5.3 y, including 78 primary, 21 implant revisions, and 15 reimplantations for recurrence. Tibial LTTBP resulted in a mean change of +8.6 o in MPTA and corrected 53% of tibias. CLGM resulted in MAD correction for 54% of limbs.Univariate analysis showed that success was best predicted by preoperative age, weight, MPTA, and MAD. Multivariate analysis identified that preoperative-MPTA/MAD and preoperative-weight<70 kg were predictive of MPTA and MAD correction, respectively. The probability of success tables are presented for reference., Conclusion: Successful correction of MPTA to age-adjusted norms following a single-event LTTBP occurred in 53% of tibias and was best predicted by preoperative-MPTA and preoperative body weight <70 kg. Comprehensive growth modulation corrected limbs in 54%. The probability of correction to age-adjusted MAD is best estimated by preoperative-MAZone 1 or 2 (MAD ≤40 mm). Limbs with preoperative-MAD>80 mm improved, but ultimately all failed to correct completely with CLGM. Osteotomy may need to be considered with these severe deformities. While modified Langenskiöld classification and medial physeal slope have been shown to predict the outcome of osteotomy, they were not predictive for LTTBP. Change in MPTA was common after physeal untethering., Level of Evidence: Level-III., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Growth modulation in late-onset tibia vara (LOTV) has been reported to yield variable results. We hypothesized that parameters of deformity severity, skeletal maturity, and body weight could predict the odds of a successful outcome., Methods: A retrospective review of tension band growth modulation for LOTV (onset ≥8 y) was performed at 7 centers. Tibial/overall limb deformity and hip/knee physeal maturity were assessed on preoperative anteroposterior standing lower-extremity digital radiographs. Tibial deformity change with first-time lateral tibial tension band plating (first LTTBP) was assessed by medial proximal tibia angle (MPTA). Effects of a growth modulation series (GMS) on overall limb alignment were assessed by mechanical tibiofemoral angle (mTFA) and included changes from implant removal, revision, reimplantation, subsequent growth, and femoral procedures during the study period. The successful outcome was defined as radiographic resolution of varus deformity or valgus overcorrection. Patient demographics, characteristics, maturity, deformity, and implant selections were assessed as outcome predictors using multiple logistic regression., Results: Fifty-four patients (76 limbs) had 84 LTTBP procedures and 29 femoral tension band procedures. For each 1-degree decrease in preoperative MPTA or 1-degree increase in preoperative mTFA the odds of their successful correction decreased by 26% in the first LTTBP and 6% by GMS, respectively, controlling for maturity. The change in odds of success for GMS assessed by mTFA was similar when controlling for weight. Closure of a proximal femoral physis decreased the odds of success for postoperative-MPTA by 91% with first LTTBP and for final-mTFA by 90% with GMS, controlling for preoperative deformity. Preoperative weight ≥100 kg decreased the odds of success for final-mTFA with GMS by 82%, controlling for preoperative mTFA. Age, sex, race/ethnicity, type of implant, and knee center peak value adjusted age (a method for bone age) were not predictive of outcome., Conclusions: Resolution of varus alignment in LOTV using first LTTBP and GMS, as quantified by MPTA and mTFA, respectively, is negatively impacted by deformity magnitude, hip physeal closure, and/or body weight ≥100 kg. The presented table, utilizing these variables, is helpful in the prediction of the outcome of the first LTTBP and GMS. Even if complete correction is not predicted, growth modulation may still be appropriate to reduce deformity in high-risk patients., Level of Evidence: Level III., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Patients should be prescribed medication based on their medical condition, without prejudice because of their race, gender, or primary language. However, previous research has shown that men are prescribed more medication than women, patients who are White are prescribed more medications than patients who are non-White, and English-speaking people are prescribed more medications than non-English-speaking patients. However, it is unclear whether these differences also occur in pediatric orthopaedic populations., Questions/purposes: We asked: (1) Was the amount of opiates prescribed at discharge associated with patient age, gender, race, or primary language? (2) Did the amount of opiates prescribed to patients at discharge change from 2010 to 2020?, Methods: In a single center, between January 2010 and December 2019, we treated 331 patients younger than 18 years surgically for upper and lower long-bone extremity fractures. Patients were considered eligible if they had a nonpathologic fracture. Femur fractures were not included. Based on these criteria, all patients were eligible. The mean age was 12 ± 4 years. The mean weight was 57 ± 33 kg. Among these patients, 76% (253 of 331) were boys and 24% (78 of 331) were girls. From the hospital discharge records, we recorded the amount of opiates prescribed at the time of discharge as measured by morphine milligram equivalents (MMEs). We examined the association of age, gender, race, primary language, weight, and year of treatment using this measurement. We determined a patient's race retrospectively by information given by their parents at time of admission. We did not attempt to contact patients to obtain more nuanced information about their racial background. These data were obtained from the electronic health record. The Wilcoxon rank sum test, t-test, or chi-square test was used to assess associations depending on the distribution of variables, as appropriate. Because opioids as measured in MMEs is zero-inflated, a two-part model analysis was used to adjust for confounding variables. One component of the model was for the probability of having any opiate prescription and another was for the mean number of opioids received. Findings were considered statistically significant if p values were < 0.05., Results: In total, 57% (189 of 331) of children were prescribed opiates at discharge after surgery for long-bone fractures. Opiate MMEs increased with patient age (r = 0.38; p < 0.01). Boys and girls showed no difference in the amounts of pain medication (adjusted odds ratio [OR] 1.38 [95% confidence interval (CI) 0.80 to 2.39]; p = 0.71; adjusted opioid difference: 0.35 MME [95% CI -51.7 to 52.4]; p = 0.99), nor were there differences between patients who were White and those who were non-White (adjusted OR 0.78 [95% CI 0.49 to 1.23]; p = 0.28; adjusted opioid difference: 21.5 MME [95% CI -19.3 to 62.4]; p = 0.30), or between patients for whom English was there primary language and those for whom English was not their primary language (adjusted OR 1.16 [95% CI 0.52 to 2.57]; p = 0.71; adjusted opioid difference: 22.7 MME [95% CI -55.7 to 101.3]; p = 0.57) when adjusted for age and weight. There was no change in opioid prescription amounts from 2010 to 2020 after adjusting for changes in patient age across years (Spearman r = -0.08; p = 0.16)., Conclusion: Fairness in opioid prescribing based on race, gender, or primary language is possible. Additional research is needed to determine what factors in our institution led to this result. We suggest that prescribers should apply consistent protocols based on factors such as weight or injury type rather than making individual decisions for each patient. This will lead to fairer opioid prescribing to patients from different race and gender groups., Level of Evidence: Level III, therapeutic study., Competing Interests: Each author certifies that there are no funding or commercial associations (consultancies, stock ownership, equity interest, patent/licensing arrangements, etc.) that might pose a conflict of interest in connection with the submitted article related to the author or any immediate family members. All ICMJE Conflict of Interest Forms for authors and Clinical Orthopaedics and Related Research® editors and board members are on file with the publication and can be viewed on request., (Copyright © 2022 by the Association of Bone and Joint Surgeons.)

Background: Despite early osteotomy, many patients with infantile tibia vara (ITV) have persistent or recurrent varus deformity and disordered growth at the medial proximal tibial physis. Our hypothesis was that lateral tibial tension band plating (LTTBP) could guide correction., Methods: A retrospective review at 6 centers of 15 patients (16 extremities) was performed of LTTBP for varus deformity following early osteotomy in ITV, diagnosed≤4years of age. Correction of deformity parameters on digital standing anteroposterior lower extremity radiographs determined outcome., Results: Twenty-two LTTBP procedures were performed at mean age of 7.5 years, including 4 revisions for implant failure and 2 reimplantations for recurrence. Single event LLTBP, improved the medial proximal tibial angle with a mean change of 13.4 degrees (0.39 degrees/month). Eleven limbs had preoperative mechanical lateral distal femoral angle (mLDFA)>90 degrees. While n degree femoral procedures were performed, at study end, 11 femurs had mechanical lateral distal femoral angle≤90°. Pretreatment, 13 extremities had mechanical axis zone (MAZone) III varus (81%) and 3 had MAZone II varus (19%). LTTBP's were able to initially correct 13 limbs to MAZone I or valgus but 4 limbs rebounded to MAZone II varus after implant removal. Final limb alignment, after all surgeries and rebound, included 9 in MAZone I, 5 in MAZone II varus and 2 in MAZone III varus. Average follow-up was 3.0 years at mean 10.7 years of age. Fifteen procedures resulted in improvement in MAZone and 7 had no change. On average, those that improved were younger (7.3 vs. 8.0 y), weighed less (45.5 kg with body mass index 26.5 kg/m 2 vs. 67.8 kg and body mass index 35.7 kg/m 2 ), had lower mechanical axis deviation (37.1 mm vs. 43.9 mm), lower medial physeal slope (61.7 vs. 68.7 degrees) and had a higher percentage of open triradiate phases (87 vs. 57%)., Conclusions: LTTBP for residual varus, after initial osteotomy in ITV, resulted in 81% of limbs initially achieving MAZone I or valgus with implant failure revisions and femoral remodeling. Rebound after implant removal reduced the corrected rate to 56%. Ninety-four percent avoided osteotomy during the study period., Level of Evidence: IV., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Proximal focal femoral deficiency is a congenital transverse deficiency in which the femur is globally smaller with a typical proximal deformity at the hip that may include distal involvement of knees, leg, and feet. Congenital femoral deficiency (CFD) describes a broader spectrum of longitudinal deficiency inclusive of proximal focal femoral deficiency. CFD may also include lateral distal femoral hypoplasia, knee cruciate ligament deficiency, rotational instability, patellar dislocation, fibular hemimelia, ray absence, and contralateral limb involvement. Treatment intends to maximize function by limb equalization and deformity correction ranging from nonsurgical management using prosthetics to amputation and may include lengthening, shortening, and complex limb reconstruction. Management decisions depend on overall severity and the patient and family's preferences and priorities. Owing to its complexity, CFD is best treated by clinicians with considerable deformity treatment experience who can help guide decision making and embark on a treatment course that will maximize the functional outcome., (Copyright © 2022 by the American Academy of Orthopaedic Surgeons.)

Background: Tension band plate and screw implants (TBI) are frequently used for temporary hemiepiphyseodeses to manage angular deformity in growing children. The reported implant breakage rate, when TBI is used for deformities in patients with Blount disease, is much higher than when used in other diagnoses. Our hypothesis is that perioperative factors can identify risks for TBI breakage., Methods: A retrospective case-control study was performed of 246 TBI procedures in 113 patients with Blount disease at 8 tertiary pediatric orthopaedic centers from 2008 to 2018. Patient demographics, age at diagnosis, weight, body mass index (BMI), radiographic deformity severity measures, location, and types of implants were studied. The outcome of implant breakage was compared with these perioperative factors using univariate logistic regression with Bonferroni correction for multiplicity to significance tests., Results: There were 30 broken implants (12%), failing at mean 1.6 years following implantation. Most failures involved the metaphyseal screws. Increased BMI was associated with increased implant breakage. Increased varus deformity was directly associated with greater implant breakage and may be a more important factor in failure for those below 7 years compared with those 8 years or above at diagnosis. There was a 50% breakage rate for TBI with solid 3.5 mm screws in Blount disease with onset 8 years or above of age. No demographic or implant factors were found to be significant., Conclusions: Breakage of TBI was associated with increased BMI and varus deformity in patients with Blount disease. Larger studies are required to determine the relative contribution and limits of each parameter. Solid 3.5 mm screws should be used with caution in TBI for late-onset Blount disease., Level of Evidence: Level III., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Anterior cruciate ligament reconstruction is the preferred treatment to anterior cruciate ligament injury. With the increase in anterior cruciate ligament injuries in both adults and skeletally immature patients comes the need for individualized anterior cruciate ligament reconstruction graft selection whether it is the type of graft (auto vs. allograft) or the harvesting site (hamstrings, iliotibial band, quadriceps, patella). Several factors need to be considered preoperatively in order to optimize the patients' recovery and restore anterior cruciate ligament strength and function. These include age and bone maturity, preoperative knee flexor/extensor strength, sport participation, patient's needs and anatomical characteristics. This paper aims at bringing evidence supporting the use of a personalized approach in graft selection for faster and more efficient return to sport and propose a theoretical framework to support the approach., (© 2022. The Author(s).)

Objectives : Epidemiology of patellar instability is not fully characterized, as some studies have produced conflicting information or been drawn from limited populations. In this study, we used the Pediatric Health Information System (PHIS) database to examine trends in admissions for patellar instability throughout the United States. We hypothesized that there would be an increasing rate of patellar instability admissions in recent years. Secondarily, we examined how demographic factors and insurance status relate to admissions for patellar instability. Methods : This study queried the Pediatric Health Information System (PHIS). Patients with ICD 9 & 10 codes 8363, 8364, 71,785, M221.0, S83.001, S83.002, S83.003, S83.004, S83.005, and S83.006, were abstracted from PHIS. We included all patients admitted with patellofemoral dislocations that were reported in the PHIS database between 1 January 2004 and 30 April 2017. The rate of patellar instability admissions was examined as well as demographic factors and insurance status. Results : An estimated total of 25,413 admissions for patellar instability were identified by the search of the PHIS database; 15,444 (60.8%) were female patients and 9,966 (39.2%) were male. The adjusted number of admissions per reporting hospital significantly increased over the years studied (r = 0.775, p < 0.001), from 14.5 admissions per reporting hospital in 2004 to 86.0 admissions per reporting hospital in 2016. Of all patellar instability admissions, 55.0% were white, 20.4% were black, 1.7% were Asian, and 22.9% were unknown race. A larger number of admissions occurred in patients with insurance status other than Medicaid in every year data were collected. Conclusions : Based on a PHIS database search, pediatric hospital admissions for patellar instability are steadily increasing. The majority of patients admitted for patellar instability are female, white, and have insurance other than Medicaid. Males admitted for patellar instability tended to be older than females admitted for the same. Level of evidence : II.

Background: Phenomena including early sport specialization and year-round training and competition have contributed to an increase in pediatric sports injuries. There has been a concomitant increase in clinical studies focusing on physically active children and adolescents. These studies include investigations of patient-reported outcome measures (PROMs). While the use of PROMs in pediatric orthopaedics has been increasing, PROMs are often inappropriately applied to study populations for whom they are not specifically validated., Purpose: The purpose of this study was to establish a comprehensive list of pediatric- and adolescent-validated PROMs and catalog their psychometric properties as a resource for clinicians and researchers., Study Design: Systematic review; Level of evidence, 4., Methods: A systematic review of articles in PubMed, Embase, and the Cochrane library was performed to identify articles developing and validating PROMs appropriate for use in pediatric sports medicine research. The inclusion criteria were as follows: age <18 years, the use of surveys and questionnaires, and the presence of sports-related injuries. The psychometric properties of included PROMs were entered into an electronic database., Results: Our search strategy identified 14,708 unique articles, among which 139 studies (0.9%) were included in the final data analysis. Forty-seven distinct PROMs were identified, as well as 160 cross-cultural adaptations. While all identified PROMs were validated in physically active young participants, only 12 (26%) were specifically created initially for active children. Thirty (64%) PROMs were health-related quality-of-life measures; 13 (28%) were psychosocial measures; and 4 (9%) were activity scales. No studies validated PROMs for use with wrist/hand injuries, and only 1 PROM each was valid for hip, back/spinal, and foot/ankle injuries in pediatric sports., Conclusion: This systematic review yielded 47 unique PROMs reliable and valid for use in pediatric and adolescent sports medicine. This list will unify clinicians and researchers in using these age-appropriate measures while identifying areas that are still in need of appropriate PROMs for young athletes.

Osteoporosis, often called a silent disease, is a systemic condition of bone as a result of loss of bone mass and deterioration of its microarchitecture. The result is weakened bone, leading to an increased risk of fragility fractures. An estimated 9 million osteoporotic fractures occur every year worldwide. However, the true incidence of osteoporotic fractures is unknown because many are undetected. Astoundingly, this epidemic equates to an osteoporotic fracture every 3 seconds. Orthopaedic surgeons need to not only treat these fractures but also understand the underlying pathogenesis and risk factors to help prevent them. The management of osteoporosis is a critical part of musculoskeletal care. We must be familiar with the tools to assess osteoporosis and the treatments available, including risks and benefits. This review article is intended to deliver a review of the vast literature and provide the orthopaedic surgeon with the essential information necessary to manage the current osteoporosis epidemic.

Data from the US Census Bureau, the Accreditation Council for Graduate Medical Education, and the American Academy of Orthopaedic Surgeons reveal that orthopedic surgery is the least diverse of any surgical specialty and that diversity within orthopedics is not improving. Considerable data from both medicine and business suggest that improving diversity within the orthopedic surgery specialty would be of significant benefit to the orthopedic surgery profession and to patients. Multiple avenues for increasing diversity exist, including large-scale pipeline programs as well as personal and institutional efforts examining biases and decision-making processes., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Twenty percent of shoulder dislocations occur in people younger than 20 years old. Management of anterior shoulder instability in young patients remains an evolving and controversial topic. Herein we review the natural history of anterior shoulder dislocation in young patients., Methods: The English-language literature was searched for studies examining the natural history of pediatric and adolescent shoulder dislocation. Recurrent dislocation was the primary outcome of interest in most studies., Results: Most studies found that recurrent instability was likely in young patients. Several systematic reviews reported the recurrence rate for young patients to be >70%. Recurrent instability was likely to cause greater damage to the joint and may result in more extensive and costly surgery., Conclusions: Most studies agree that recurrent anterior shoulder instability is likely in young patients. Some authors advocate for consideration of early surgery in this high-risk population.

Background: Performance on the Orthopaedic In-training Examination (OITE) has been correlated with performance on the written portion of the American Board of Orthopaedic Surgery examination. Herein we sought to discover whether adding a regular pediatric didactic lecture improved residents' performance on the OITE's pediatric domain., Methods: In 2012, a didactic lecture series was started in the University of Pittsburgh Medical Center (UPMC) Hamot Orthopaedic Residency Program (Hamot). This includes all topics in pediatric orthopaedic surgery and has teaching faculty present, and occurs weekly with all residents attending. A neighboring program [UMPC Pittsburgh (Pitt)] shares in these conferences, but only during their pediatric rotation. We sought to determine the effectiveness of the conference by comparing the historic scores from each program on the pediatric domain of the OITE examination to scores after the institution of the conference, and by comparing the 2 programs' scores., Results: Both programs demonstrated improvement in OITE scores. In 2008, the mean examination score was 19.6±4.3 (11.0 to 30.0), and the mean percentile was 57.7±12.6 (32.0 to 88.0); in 2014, the mean examination score was 23.5±4.2 (14.0 to 33.0) and the mean percentile was 67.1±12.1 (40.0 to 94.0). OITE scores and percentiles improved with post graduate year (P<0.0001). Compared with the preconference years, Hamot residents answered 3.99 more questions correctly (P<0.0001) and Pitt residents answered 2.93 more questions correctly (P<0.0001). Before the conference, site was not a predictor of OITE score (P=0.06) or percentile (P=0.08); there was no significant difference found between the mean scores per program. However, in the postconference years, site did predict OITE scores. Controlling for year in training, Hamot residents scored higher on the OITE (2.3 points higher, P=0.003) and had higher percentiles (0.07 higher, P=0.004) than Pitt residents during the postconference years., Conclusions: This study suggests that adding a didactic pediatric lecture improved residents' scores on the OITE and indirectly suggests that more frequent attendance is associated with better scores., Level of Evidence: Level III-retrospective case-control study.

Acetylcholine regulates memory encoding and retrieval by inducing the hippocampus to switch between pattern separation and pattern completion modes. However, both processes can introduce significant variations in the level of network activity and potentially cause a seizure-like spread of excitation. Thus, mechanisms that keep network excitation within certain bounds are necessary to prevent such instability. We developed a biologically realistic computational model of the hippocampus to investigate potential intrinsic mechanisms that might stabilize the network dynamics during encoding and retrieval. The model was developed by matching experimental data, including neuronal behavior, synaptic current dynamics, network spatial connectivity patterns, and short-term synaptic plasticity. Furthermore, it was constrained to perform pattern completion and separation under the effects of acetylcholine. The model was then used to investigate the role of short-term synaptic depression at the recurrent synapses in CA3, and inhibition by basket cell (BC) interneurons and oriens lacunosum-moleculare (OLM) interneurons in stabilizing these processes. Results showed that when CA3 was considered in isolation, inhibition solely by BCs was not sufficient to control instability. However, both inhibition by OLM cells and short-term depression at the recurrent CA3 connections stabilized the network activity. In the larger network including the dentate gyrus, the model suggested that OLM inhibition could control the network during high cholinergic levels while depressing synapses at the recurrent CA3 connections were important during low cholinergic states. Our results demonstrate that short-term plasticity is a critical property of the network that enhances its robustness. Furthermore, simulations suggested that the low and high cholinergic states can each produce runaway excitation through unique mechanisms and different pathologies. Future studies aimed at elucidating the circuit mechanisms of epilepsy could benefit from considering the two modulatory states separately., (© 2014 Wiley Periodicals, Inc.)

Background: Debate regarding the optimal initial treatment for anterior cruciate ligament (ACL) injuries in children and adolescents has not resulted in a clear consensus for initial nonoperative treatment or operative reconstruction., Hypothesis/purpose: The purpose of this meta-analysis was to systematically analyze aggregated data from the literature to determine if a benefit exists for either nonoperative or early operative treatment for ACL injuries in the pediatric patient. The hypothesis was that combined results would favor early operative reconstruction with respect to posttreatment episodes of instability/pathological laxity, symptomatic meniscal tears, clinical outcome scores, and return to activity., Study Design: Meta-analysis., Methods: A literature selection process included the extraction of data on the following clinical variables: symptomatic meniscal tears, return to activities, clinical outcome scores, return to the operating room, and posttreatment instability/pathological laxity. A symptomatic meniscal tear was defined as occurring after the initial presentation, limiting activity, and requiring further treatment. Instability/pathological laxity was defined for the sake of this study as having an episode of giving way, a grade ≥2 Lachman/pivot-shift test result, or a side-to-side difference of >4 mm as measured by the KT-1000 arthrometer. All studies were evaluated using a formal study quality analysis. Meta-analysis was conducted for aggregated data in each category., Results: Six studies (217 patients) comparing operative to nonoperative treatment and 5 studies (353 patients) comparing early to delayed reconstruction were identified. Three studies reported posttreatment instability/pathological laxity; 13.6% of patients after operative treatment experienced instability/pathological laxity compared with 75% of patients after nonoperative treatment (P < .01). Two studies reported symptomatic meniscal tears; patients were over 12 times more likely to have a medial meniscal tear after nonoperative treatment than after operative treatment (35.4% vs 3.9%, respectively; P = .02). A significant difference in scores between groups was noted in 1 of 2 studies reporting International Knee Documentation Committee (IKDC) scores (P = .002) and in 1 of 2 studies reporting Tegner scores (P = .007). Two studies reported return to activity; none of the patients in the nonoperative groups returned to their previous level of play compared with 85.7% of patients in the operative groups (P < .01). Study quality analysis revealed that the majority of the studies were inconsistent in reporting outcomes., Conclusion: Meta-analysis revealed multiple trends that favor early surgical stabilization over nonoperative or delayed treatment. Patients after nonoperative and delayed treatment experienced more instability/pathological laxity and inability to return to previous activity levels than did patients treated with early surgical stabilization., (© 2013 The Author(s).)

Forearm fractures occur commonly in children; however, there is still uncertainty on what leads to conversion from conservative to operative management. Patients who initially underwent closed reduction and casting for diaphyseal forearm fractures were evaluated for predictors of conversion to operative management. We found that the 20 of 124 (16%) patients in whom there was conversion to operative management were significantly older (11.1 vs. 5.7 nonoperative), had less angulation in the anterior-posterior (or coronal) plane (20.2 vs. 12.8° for the radius, 17.5 vs. 7.8° for the ulna), had a more proximal ulnar fracture location, and had more translated or shortened radius fractures.

Glutathionylation is a posttranslational modification that results in the formation of a mixed disulfide between glutathione and the thiol group of a protein cysteine residue. Glutathionylation of proteins occurs via both nonenzymatic mechanisms involving thiol/disulfide exchange and enzyme-mediated reactions. Protein glutathionylation is observed in response to oxidative or nitrosative stress and is redox-dependent, being readily reversible under reducing conditions. Such findings suggest that glutathionylation plays an important role in mediating redox-sensitive signaling. Indeed, glutathionylation can affect protein function by altering activity, protein-protein interactions, and ligand binding. Glutathionylation may also serve to prevent cysteine residues from undergoing irreversible oxidative modification. Thus, determining the ability of a given protein to become glutathionylated can provide insight into its redox regulation and putative role in dictating cellular response to oxidative and nitrosative stress. Methods to measure protein glutathionylation using immunoblotting and mass spectrometry are described., (Copyright © 2013 John Wiley & Sons, Inc.)

The antioxidant glutathione (GSH) plays a critical role in maintaining intracellular redox homeostasis but in tumors the GSH biosynthetic pathway is often dysregulated, contributing to tumor resistance to radiation and chemotherapy. Glutamate-cysteine ligase (GCL) catalyzes the first and rate-limiting reaction in GSH synthesis, and enzyme function is controlled by GSH feedback inhibition or by transcriptional upregulation of the catalytic (GCLC) and modifier (GCLM) subunits. However, it has recently been reported that the activity of GCLC and the formation of GCL can be modified by reactive aldehyde products derived from lipid peroxidation. Due to the susceptibility of GCLC to posttranslational modifications by reactive aldehydes, we examined the potential for 2-deoxy-D-ribose (2dDR) to glycate GCLC and regulate enzyme activity and GCL formation. 2dDR was found to directly modify both GCLC and GCLM in vitro, resulting in a significant inhibition of GCLC and GCL enzyme activity without altering substrate affinity or feedback inhibition. 2dDR-mediated glycation also inhibited GCL subunit heterodimerization and formation of the GCL holoenzyme complex while not causing dissociation of pre-formed holoenzyme. This PTM could be of particular importance in glioblastoma (GBM) where intratumoral necrosis provides an abundance of thymidine, which can be metabolized by thymidine phosphorylase (TP) to form 2dDR. TP is expressed at high levels in human GBM tumors and shRNA knockdown of TP in U87 GBM cells results in a significant increase in cellular GCL enzymatic activity.

Anterior cruciate ligament injuries in the pediatric population have been increasing in recent years. While reconstruction can often provide the best chance of restoring stability and preventing degenerative joint disease, the skeletally immature patient with open physes can represent a treatment challenge to the orthopaedic surgeon. Here, we present a technique developed by the senior author that uses two-dimensional fluoroscopy, obviating the need for computed tomography imaging. Intraoperative technical details of this procedure are highlighted. This technique allows the orthopaedic surgeon to reconstruct the ligament in a physeal-sparing manner without the need for intraoperative computed tomography scanning, and with less risk of radiation exposure.

Supracondylar fractures of the humerus are exceedingly common in pediatric patients but may present treatment challenges when complicated by neurovascular compromise. Patients presenting with poor perfusion should be treated with urgent reduction because this is a limb-threatening emergency. If perfusion does not improve, or if a previously perfused extremity loses perfusion after reduction, arterial exploration is warranted. Controversy exists over whether to observe or explore a reduced, perfused, but pulseless extremity with a supracondylar fracture. Minimal management requires that these injuries be carefully monitored for 48 hours for loss of perfusion or the development of compartment syndrome. In general, nerve injuries accompanying supracondylar fractures of the humerus are neurapraxias and may be treated conservatively; however, nerve palsy with accompanying pulselessness warrants immediate exploration. Patients should be treated more urgently if excessive swelling, antecubital ecchymosis, skin puckering, an absent pulse, or fractures in the same limb are present. If a hand is not perfused or compartments are firm, emergent treatment should be considered.

Forearm fractures are common injuries in the pediatric population. Successful treatment of these fractures should result in complication-free functional pronosupination. Traditionally, these have been treated with closed reduction and casting, but the last several decades have seen a dramatic increase in the operative treatment of these fractures. However, little high-level evidence exists to guide management. The data from the limited set of studies available suggest that closed treatment does usually result in satisfactory outcomes, particularly in younger patients; operative fixation is usually successful as well but comes with a significantly increased complication rate. The ideal study to aid in evidence-based decision-making for pediatric forearm fractures would be a randomly controlled trial comparing closed reduction and casting versus intramedullary nailing versus plating; in children ranging from 8 years old to skeletally mature; with closed forearm fractures, complete or greenstick with >20 degrees of angulation; with a minimum of 5 years of follow-up (or to maturity); with the primary outcome defined as final pronation and supination; using an validated functional outcome tool; and precisely defining the complications from each treatment.

Chemotherapy is central to the current treatment modality for primary human brain tumors, but despite high-dose and intensive treatment regimens there has been little improvement in patient outcome. The development of tumor chemoresistance has been proposed as a major contributor to this lack of response. While there have been some improvements in our understanding of the molecular mechanisms underlying brain tumor drug resistance over the past decade, the contribution of glutathione (GSH) and the GSH-related enzymes to drug resistance in brain tumors have been largely overlooked. GSH constitutes a major antioxidant defense system in the brain and together with the GSH-related enzymes plays an important role in protecting cells against free radical damage and dictating tumor cell response to adjuvant cancer therapies, including irradiation and chemotherapy. Glutamate cysteine ligase (GCL), glutathione synthetase (GS), glutathione peroxidase (GPx), glutathione reductase (GR), glutathione-S-transferases (GST), and GSH complex export transporters (GS-X pumps) are major components of the GSH-dependent enzyme system that function in a dynamic cascade to maintain redox homeostasis. In many tumors, the GSH system is often dysregulated, resulting in a more drug resistant phenotype. This is commonly associated with GST-mediated GSH conjugation of various anticancer agents leading to the formation of less toxic GSH-drug complexes, which can be readily exported from the cell. Advances in our understanding of the mechanisms of drug resistance and patient selection based on biomarker profiles will be crucial to adapt therapeutic strategies and improve outcomes for patients with primary malignant brain tumors., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Chalcones continue to attract considerable interest due to their anti-inflammatory and antiangiogenic properties. We recently reported the ability of 2',5'-dihydroxychalcone (2',5'-DHC) to induce both breast cancer resistance protein-mediated export of glutathione (GSH) and c-Jun N-terminal kinase-mediated increased intracellular GSH levels. Herein, we report a structure-activity relationship study of a series of 30 synthetic chalcone derivatives with hydroxyl, methoxyl, and halogen (F and Cl) substituents and their ability to increase intracellular GSH levels. This effect was drastically improved with one or two electrowithdrawing groups on phenyl ring B and up to three methoxyl and/or hydroxyl groups on phenyl ring A. The optimal structure, 2-chloro-4',6'-dimethoxy-2'-hydroxychalcone, induced both a potent NF-E2-related factor 2-mediated transcriptional response and an increased formation of glutamate cysteine ligase holoenzyme, as shown using a human breast cancer cell line stably expressing a luciferase reporter gene driven by antioxidant response elements.

Alcoholic liver disease (ALD) is a primary cause of morbidity and mortality in the United States and constitutes a significant socioeconomic burden. Previous work has implicated oxidative stress and endoplasmic reticulum (ER) stress in the etiology of ALD; however, the complex and interrelated nature of these cellular responses presently confounds our understanding of ethanol-induced hepatopathy. In this paper, we assessed the pathological contribution of oxidative stress and ER stress in a time-course mouse model of early-stage ALD. Ethanol-treated mice exhibited significant hepatic panlobular steatosis and elevated plasma ALT values compared to isocaloric controls. Oxidative stress was observed in the ethanol-treated animals through a significant increase in hepatic TBARS and immunohistochemical staining of 4-HNE-modified proteins. Hepatic glutathione (GSH) levels were significantly decreased as a consequence of decreased CBS activity, increased GSH utilization, and increased protein glutathionylation. At the same time, immunoblot analysis of the PERK, IRE1α, ATF6, and SREBP pathways reveals no significant role for these UPR pathways in the etiology of hepatic steatosis associated with early-stage ALD. Collectively, our results indicate a primary pathogenic role for oxidative stress in the early initiating stages of ALD that precedes the involvement of the ER stress response.

Hydroxychalcones are naturally occurring compounds that continue to attract considerable interest because of their anti-inflammatory and antiangiogenic properties. They have been reported to inhibit the synthesis of the inducible nitric oxide synthase and to induce the expression of heme oxygenase-1. This study examines the mechanisms by which 2',5'-dihydroxychalcone (2',5'-DHC) induces an increase in cellular glutathione (GSH) levels using a cell line stably expressing a luciferase reporter gene driven by antioxidant-response elements (MCF-7/AREc32). The 2',5'-DHC-induced increase in cellular GSH levels was partially inhibited by the catalytic antioxidant MnTDE-1,3-IP(5+), suggesting that reactive oxygen species (ROS) mediate the antioxidant adaptive response. 2',5'-DHC treatment induced phosphorylation of the c-Jun N-terminal kinase (JNK) pathway, which was also inhibited by MnTDE-1,3-IP(5+). These findings suggest a ROS-dependent activation of the AP-1 transcriptional response. However, whereas 2',5'-DHC triggered the NF-E2-related factor 2 (Nrf2) transcriptional response, cotreatment with MnTDE-1,3-IP(5+) did not decrease 2',5'-DHC-induced Nrf2/ARE activity, showing that this pathway is not dependent on ROS. Moreover, pharmacological inhibitors of mitogen-activated protein kinase (MAPK) pathways showed a role for JNK and p38MAPK in mediating the 2',5'-DHC-induced Nrf2 response. These findings suggest that the 2',5'-DHC-induced increase in GSH levels results from a combination of ROS-dependent and ROS-independent pathways., (Copyright © 2011 Elsevier Inc. All rights reserved.)

4-Hydroxy-2-nonenal (4-HNE) is a lipid peroxidation product formed during oxidative stress that can alter protein function via adduction of nucleophilic amino acid residues. 4-HNE detoxification occurs mainly via glutathione (GSH) conjugation and transporter-mediated efflux. This results in a net loss of cellular GSH, and restoration of GSH homeostasis requires de novo GSH biosynthesis. The rate-limiting step in GSH biosynthesis is catalyzed by glutamate-cysteine ligase (GCL), a heterodimeric holoenzyme composed of a catalytic (GCLC) and a modulatory (GCLM) subunit. The relative levels of the GCL subunits are a major determinant of cellular GSH biosynthetic capacity and 4-HNE induces the expression of both GCL subunits. In this study, we demonstrate that 4-HNE can alter GCL holoenzyme formation and activity via direct posttranslational modification of the GCL subunits in vitro. 4-HNE directly modified Cys553 of GCLC and Cys35 of GCLM in vitro, which significantly increased monomeric GCLC enzymatic activity, but reduced GCL holoenzyme activity and formation of the GCL holoenzyme complex. In silico molecular modeling studies also indicate these residues are likely to be functionally relevant. Within a cellular context, this novel posttranslational regulation of GCL activity could significantly affect cellular GSH homeostasis and GSH-dependent detoxification during periods of oxidative stress., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The underlying membrane potential oscillation of both forced and endogenous slow-wave bursting cells affects the number of spikes per burst, which in turn affects outputs downstream. We use a biophysical model of a class of slow-wave bursting cells with six active currents to investigate and generalize correlations among maximal current conductances that might generate and preserve its underlying oscillation. We propose three phases for the underlying oscillation for this class of cells: generation, maintenance, and termination and suggest that different current modules coregulate to preserve the characteristics of each phase. Coregulation of I(Burst) and I(A) currents within distinct boundaries maintains the dynamics during the generation phase. Similarly, coregulation of I(CaT) and I(Kd) maintains the peak and duration of the underlying oscillation, whereas the calcium-activated I(KCa) ensures appropriate termination of the oscillation and adjusts the duration independent of peak.

Similar activity patterns at both neuron and network levels can arise from different combinations of membrane and synaptic conductance values. A strategy by which neurons may preserve their electrical output is via cell type-dependent balances of inward and outward currents. Measurements of mRNA transcripts that encode ion channel proteins within motor neurons in the crustacean cardiac ganglion recently revealed correlations between certain channel types. To determine whether balances of intrinsic currents potentially resulting from such correlations preserve certain electrical cell outputs, we developed a nominal biophysical model of the crustacean cardiac ganglion using biological data. Predictions from the nominal model showed that coregulation of ionic currents may preserve the key characteristics of motor neuron activity. We then developed a methodology of sampling a multidimensional parameter space to select an appropriate model set for meaningful comparison with variations in correlations seen in biological datasets.

Glutamate cysteine ligase (GCL) catalyzes the rate-limiting step in the formation of the cellular antioxidant glutathione (GSH). The GCL holoenzyme consists of two separately coded proteins, a catalytic subunit (GCLC) and a modifier subunit (GCLM). Both GCLC and GLCM are controlled transcriptionally by a variety of cellular stimuli, including oxidative stress. This study addresses post-translational control of GCL activity, which increased rapidly in human lymphocytes following oxidative stress. Activation of GCL occurred within minutes of treatment and without any change in GCL protein levels and coincided with an increase in the proportion of GCLC in the holoenzyme form. Likewise, GCLM shifted from the monomeric form to holoenzyme and higher molecular weight species. Normal rat tissues also showed a distribution of monomeric and higher molecular weight forms. Neither GCL activation, nor the formation of holoenzyme, required a covalent intermolecular disulfide bridge between GCLC and GCLM. However, in immunoprecipitation studies, a neutralizing epitope associated with enzymatic activity was protected following cellular oxidative stress. Thus, the N-terminal portion of GCLC may undergo a change that stabilizes the GCL holoenzyme. Our results suggest that a dynamic equilibrium exists between low and high activity forms of GCL and is altered by transient oxidative stress. This provides a mechanism for the rapid post-translational activation of GCL and maintenance of cellular GSH homeostasis.

Wegener's granulomatosis (WG) is a life-threatening autoimmune vasculitis that affects lungs, kidneys and other organs. A hallmark of WG is the presence of classic anti-neutrophil cytoplasmic antibodies (c-ANCA) against self-proteinase 3 (PR3). Little is known about the role of these antibodies and PR3-specific immune responses in disease development. In this study, we demonstrate that PR3-specific autoimmune responses are pathogenic in non-obese diabetic (NOD) mice with an impaired regulatory arm of the immune response. Immunization of autoimmunity prone NOD mice with rmPR3 (recombinant mouse PR3) in complete Freund's adjuvant (CFA) resulted in high levels of c-ANCA, without detectable disease development. However, when splenocytes from these immunized mice were transferred into immunodeficient NOD-severe combined immunodeficiency (SCID) mice, the recipient mice developed vasculitis and severe segmental and necrotizing glomerulonephritis. No disease developed in NOD-SCID mice that received splenocytes from the CFA-alone-immunized donors (controls), indicating that disease development depends upon PR3-specific immune responses. In contrast to the pathology observed in NOD-SCID mice, no disease was observed when splenocytes from rmPR3-immunized C57BL/6 mice were transferred into immunodeficient C57BL/6-RAG-1(-/-) mice, suggesting that complex and probably multi-genetic factors play a role in the regulation of disease development.

Trivalent arsenite (As(3+)) is a known human carcinogen capable of inducing both cellular transformation and apoptotic cell death by mechanisms involving the production of reactive oxygen species. The tripeptide antioxidant glutathione (GSH) constitutes a vital cellular defense mechanism against oxidative stress. While intracellular levels of GSH are an important determinant of cellular susceptibility to undergo apoptotic cell death, it is not known whether cellular GSH biosynthetic capacity per se regulates As(3+)-induced apoptosis. The rate-limiting enzyme in GSH biosynthesis is glutamate cysteine ligase (GCL), a heterodimeric holoenzyme composed of a catalytic (GCLC) and a modifier (GCLM) subunit. To determine whether increased GSH biosynthetic capacity enhanced cellular resistance to As(3+)-induced apoptotic cell death, we utilized a mouse liver hepatoma (Hepa-1c1c7) cell line stably overexpressing both GCLC and GCLM. Overexpression of the GCL subunits increased GCL holoenzyme formation and activity and inhibited As(3+)-induced apoptosis. This cytoprotective effect was associated with a decrease in As(3+)-induced caspase activation, cleavage of caspase substrates and translocation of cytochrome c to the cytoplasm. In aggregate, these findings demonstrate that enhanced GSH biosynthetic capacity promotes resistance to As(3+)-induced apoptosis by preventing mitochondrial dysfunction and cytochrome c release and highlight the role of the GSH antioxidant defense system in dictating hepatocyte sensitivity to As(3+)-induced apoptotic cell death., (Copyright 2009 Elsevier Ireland Ltd. All rights reserved.)

The glutathione (GSH) antioxidant defense system plays a central role in protecting mammalian cells against oxidative injury. Glutamate cysteine ligase (GCL) is the rate-limiting enzyme in GSH biosynthesis and is a heterodimeric holoenzyme composed of catalytic (GCLC) and modifier (GCLM) subunits. As a means of assessing the cytoprotective effects of enhanced GSH biosynthetic capacity, we have developed a protein transduction approach whereby recombinant GCL protein can be rapidly and directly transferred into cells when coupled to the HIV TAT protein transduction domain. Bacterial expression vectors encoding TAT fusion proteins of both GCL subunits were generated and recombinant fusion proteins were synthesized and purified to near homogeneity. The TAT-GCL fusion proteins were capable of heterodimerization and formation of functional GCL holoenzyme in vitro. Exposure of Hepa-1c1c7 cells to the TAT-GCL fusion proteins resulted in the time- and dose-dependent transduction of both GCL subunits and increased cellular GCL activity and GSH levels. A heterodimerization-competent, enzymatically deficient GCLC-TAT mutant was also generated in an attempt to create a dominant-negative suppressor of GCL. Transduction of cells with a catalytically inactive GCLC(E103A)-TAT mutant decreased cellular GCL activity in a dose-dependent manner. TAT-mediated manipulation of cellular GCL activity was also functionally relevant as transduction with wild-type GCLC(WT)-TAT or mutant GCLC(E103A)-TAT conferred protection or enhanced sensitivity to H(2)O(2)-induced cell death, respectively. These findings demonstrate that TAT-mediated transduction of wild-type or dominant-inhibitory mutants of the GCL subunits is a viable means of manipulating cellular GCL activity to assess the effects of altered GSH biosynthetic capacity., (Copyright 2009 Elsevier Inc. All rights reserved.)

Trivalent arsenite (As(3+)) is a known human carcinogen that is also capable of inducing apoptotic cell death. Increased production of reactive oxygen species is thought to contribute to both the carcinogenic and the cytotoxic effects of As(3+). Glutathione (GSH) constitutes a vital cellular defense mechanism against oxidative stress. The rate-limiting enzyme in GSH biosynthesis is glutamate-cysteine ligase (GCL), a heterodimeric holoenzyme composed of a catalytic (GCLC) and a modifier (GCLM) subunit. In this study, we demonstrate that As(3+) coordinately upregulates Gclc and Gclm mRNA levels in a murine hepatocyte cell line resulting in increased GCL subunit protein expression, holoenzyme formation, and activity. As(3+) increased the rate of transcription of both the Gclm and the Gclc genes and induced the posttranscriptional stabilization of Gclm mRNA. The antioxidant N-acetylcysteine abolished As(3+)-induced Gclc expression and attenuated induction of Gclm. As(3+) induction of Gclc and Gclm was also differentially regulated by the MAPK signaling pathways and occurred independent of the Nrf1/2 transcription factors. These findings demonstrate that distinct transcriptional and posttranscriptional mechanisms mediate the coordinate induction of the Gclc and Gclm subunits of GCL in response to As(3+) and highlight the potential importance of the GSH antioxidant defense system in regulating As(3+)-induced responses in hepatocytes.

Glutathione (GSH) is a tripeptide composed of glutamate, cysteine, and glycine. The first and rate-limiting step in GSH synthesis is catalyzed by glutamate cysteine ligase (GCL, previously known as gamma-glutamylcysteine synthetase). GCL is a heterodimeric protein composed of catalytic (GCLC) and modifier (GCLM) subunits that are expressed from different genes. GCLC catalyzes a unique gamma-carboxyl linkage from glutamate to cysteine and requires ATP and Mg(++) as cofactors in this reaction. GCLM increases the V(max) and K(cat) of GCLC, decreases the K(m) for glutamate and ATP, and increases the K(i) for GSH-mediated feedback inhibition of GCL. While post-translational modifications of GCLC (e.g. phosphorylation, myristoylation, caspase-mediated cleavage) have modest effects on GCL activity, oxidative stress dramatically affects GCL holoenzyme formation and activity. Pyridine nucleotides can also modulate GCL activity in some species. Variability in GCL expression is associated with several disease phenotypes and transgenic mouse and rat models promise to be highly useful for investigating the relationships between GCL activity, GSH synthesis, and disease in humans.

Neutrophils are the primary inflammatory cell in smokers' lungs, but little is known about the ability of cigarette smoke to modulate neutrophil function. Neutrophils undergo caspase-3-dependent spontaneous, as well as phagocytosis-induced, apoptosis. This study investigated the ability of cigarette smoke extract (CSE) to alter neutrophil caspase-3 activity, apoptosis, and phagocytosis. CSE treatment resulted in a dramatic suppression of neutrophil caspase-3-like activity, which correlated with reduced cleavage of glutamate-L-cysteine ligase catalytic subunit, a known target of active caspase-3. CSE did not affect procaspase-3 processing to its active fragment, suggesting a direct effect of CSE on active caspase-3. Consistent with this, CSE inhibited active recombinant caspase-3 activity, which was abolished by dithiothreitol, suggesting a redox-sensitive mechanism. CSE-induced suppression of caspase-3 activity did not alter spontaneous apoptosis but did impair phagocytic activity. Since CSE treatment resulted in profound suppression of caspase-3 activity but did not alter apoptosis, the possibility of a threshold level of caspase-3 activity was investigated. CSE reduced caspase-3 activity in a concentration-dependent manner. Despite near complete suppression of caspase-3 activity, spontaneous apoptosis was not altered. Conversely, treatment with the pan-caspase inhibitor, Z-Val-Ala-Asp-fluoromethylketone, reduced spontaneous apoptosis. These data demonstrate that CSE does not suppress caspase-3 activity below a threshold level to prevent spontaneous apoptosis, but the level of inhibition is sufficient to impair neutrophil phagocytic activity. These divergent functions of caspase-3 may contribute to the persistence of neutrophils in the lungs of smokers, as well as be a factor in their higher incidence of community-acquired pneumonia.

Background: The objective of this study was to determine the impact of malignant pleural effusions on survival in patients with optimally debulked, advanced epithelial ovarian carcinoma., Methods: The authors conducted a retrospective review of all patients with advanced epithelial ovarian carcinoma who underwent optimal primary cytoreduction at their institution between January 1987 and August 2000. Survival rates were compared between patients with optimally debulked Stage IIIC epithelial ovarian carcinoma and patients with optimally debulked Stage IV epithelial ovarian carcinoma (according to the International Federation of Gynecology and Obstetrics [FIGO] staging system) based on cytology-proven malignant pleural effusions., Results: Ninety-nine patients were identified, and 97 of those patients were evaluable. The group with Stage IIIC disease included 76 patients, and the group with Stage IV disease included 21 patients. The median age at diagnosis was 55 years (range, 26-88 years). The majority of patients received platinum-based chemotherapy after undergoing optimal primary cytoreduction. Age, tumor grade and histology, and the percentage of patients with ascites were similar in the two groups. The median survival rate was 58 months for patients who had Stage IIIC disease and 30 months for patients who had Stage IV disease (P = 0.016)., Conclusions: Although both groups underwent optimal cytoreduction in the abdomen/pelvis and were treated in a similar fashion, the median survival rate of patients with malignant pleural effusions was significantly shorter than the survival of patients without effusions. Many factors that led to or were manifested by pleural effusions, such as undetected bulky residual intrathoracic disease, may have been the cause for this survival difference. In the patients with effusions, one or more of these contributing factors may have led to the observed decreased survival rate, warranting further investigation., (Copyright 2005 American Cancer Society.)

The catalytic subunit of glutamylcysteine ligase (GCLC) primarily regulates de novo synthesis of glutathione (GSH) in mammalian cells and is central to the antioxidant capacity of the cell. However, GCLC expression in pancreatic islets has not been previously examined. We designed experiments to ascertain whether GCLC is normally expressed in islets and whether it is up-regulated by interleukin-1 beta (IL-1 beta). GCLC expression levels were intermediate compared with other metabolic tissues (kidney, liver, muscle, fat, and lung). IL-1 beta up-regulated GCLC expression (10 ng/ml IL-1 beta, 3.76 +/- 0.86; 100 ng/ml IL-1 beta, 4.22 +/- 0.68-fold control) via the p38 form of mitogen-activated protein kinase and NF kappa B and also increased reactive oxygen species levels (10 ng/ml IL-1 beta, 5.41 +/- 1.8-fold control). This was accompanied by an increase in intraislet GSH/GSSG ratio (control, 7.1 +/- 0.1; 10 ng/ml IL-1 beta, 8.0 +/- 0.5; 100 ng/ml IL-1 beta, 8.2 +/- 0.5-fold control; p < 0.05). To determine whether overexpression of GCLC increases the antioxidant capacity of the islet and prevents the adverse effects of IL-1 beta on glucose-induced insulin secretion, islets were infected with an adenovirus encoding GCLC. IL-1 beta significantly decreased glucose-stimulated insulin secretion (control, 123.8 +/- 17.7; IL-1 beta, 40.2 +/- 3.9 microunits/ml insulin/islet). GCLC overexpression increased intraislet GSH levels and partially prevented the decrease in glucose-stimulated insulin secretion caused by IL-1 beta. These data provide the first report of GCLC expression in the islet and demonstrate that adenoviral overexpression of GCLC increases intracellular GSH levels and protects the beta cell from the adverse effects of IL-1 beta.

Objective: The purpose of this study was to analyze our initial 10-year experience with laparoscopy and to determine risk factors for complications and conversions to laparotomy for technical difficulty., Study Design: We reviewed the charts of all laparoscopic procedures from January 1991 through December 2000 and divided the procedures into 4 levels on the basis of the degree of difficulty: level I, diagnostic; level II, procedures on the uterus and/or adnexa; level III, second look operations for malignancy; and level IV, lymphadenectomies/other complex procedures. Complications were graded from 1 (mild) to 5 (death). Standard univariate and multivariate analyses were performed., Results: We identified 1451 evaluable procedures. The number of complications was as follows: grades 1 to 5, 129 complications (9%); grades 3 to 5, 36 complications (2.5%). On multivariate analysis, older age ( P = .03), previous radiation ( P = .03), and malignancy ( P = .006) were associated with an increased risk of complications grades 3 to 5. Complication rates for grades 3 to 5 for patients with malignancy versus benign disease was 4% versus 1%, respectively. Technical difficulty led to conversion to laparotomy in 105 cases (7%). Previous abdominal surgery ( P < .001) significantly increased the rate of conversion to laparotomy; more complex, higher procedure levels were associated with a significant decrease in conversions ( P = .005)., Conclusion: Both simple and complex laparoscopic procedures can be performed by a gynecologic oncology service with a low rate of complications and conversions to laparotomy. Older age, malignancy, previous radiation therapy, and previous abdominal surgery were identified as significant risk factors for complications and/or conversion and should be taken into account in patient selection, preoperative counseling, and surgical planning.

Objective: To determine the impact of the incorporation of extensive upper abdominal debulking procedures on the rates of optimal primary cytoreduction and complications in stages IIIC and IV epithelial ovarian, fallopian tube, and primary peritoneal carcinomas., Methods: Two groups of patients were identified for comparison. Group 1, the control group, consisted of 70 consecutive patients who underwent "standard" primary cytoreductive surgery before May 2000. Group 2, the study group, was composed of 70 consecutive patients who underwent surgery after January 2001, during which time, a more comprehensive debulking of upper abdominal disease was used, including diaphragm stripping/resection, splenectomy, distal pancreatectomy, liver resection, resection of porta hepatis tumor, and cholecystectomy., Results: The median age of the entire cohort was 60 years (range, 36-88 years). The majority had stage IIIC disease (86%) and serous histology (76%). Optimal cytoreduction (residual disease

Glutathione (GSH) is important in free radical scavenging, maintaining cellular redox status, and regulating cell survival in response to a wide variety of toxicants. The rate-limiting enzyme in GSH synthesis is glutamate-cysteine ligase (GCL), which is composed of catalytic (GCLC) and modifier (GCLM) subunits. To determine whether increased GSH biosynthetic capacity enhances cellular resistance to tumor necrosis factor-alpha- (TNF-alpha-) induced apoptotic cell death, we have established several mouse liver hepatoma (Hepa-1) cell lines overexpressing GCLC and/or GCLM. Cells overexpressing GCLC alone exhibit modest increases in GCL activity, while cells overexpressing both subunits have large increases in GCL activity. Importantly, cells overexpressing both GCL subunits exhibit increased resistance to TNF-induced apoptosis as judged by a loss of redox potential; mitochondrial membrane potential; translocation of cytochrome c to the cytoplasm; and activation of caspase-3, caspase-8, and caspase-9. Analysis of the effects of TNF on these parameters indicates that maintaining mitochondrial integrity mediates this protective effect in GCL-overexpressing cells.

Glutamate cysteine ligase (GCL), composed of a catalytic (GCLC) and modulatory (GCLM) subunit, catalyzes the first step of glutathione (GSH) biosynthesis. Using 4-hydroxy-2-nonenal (4HNE), 2,3-dimethoxy-1,4-naphthoquinone (DMNQ), and tertiary-butylhydroquinone (tBHQ) as models of oxidative stress which are known to work through different mechanisms, we measured changes in cellular GSH, GCL mRNA, and GCL protein. 4HNE and tBHQ treatments increased cellular GSH levels, while DMNQ exposure depleted GSH. Furthermore, changes in the two GCL mRNAs largely paralleled changes in the GCL proteins; however, the magnitudes differed, suggesting some form of translational control. The molar ratio of GCLC:GCLM ranged from 3:1 to 17:1 in control human bronchial epithelial (HBE1) cells and all treatments further increased this ratio. Data from several mouse tissues show molar ratios of GCLC:GCLM that range from 1:1 to 10:1 in support of these findings. These data demonstrate that alterations in cellular GSH are clearly correlated with GCLC to a greater extent than GCLM. Surprisingly, both control HBE1 cells and some mouse tissues have more GCLC than GCLM and GCLM increases to a much lesser extent than GCLC, suggesting that the regulatory role of GCLM is minimal under physiologically relevant conditions of oxidative stress.

TGFbeta1-induced hepatocyte apoptosis involves the production of reactive oxygen species. An effective cellular defense mechanism against oxidative stress is the tripeptide glutathione (GSH), and the rate-limiting step in GSH biosynthesis is catalyzed by the heterodimeric holoenzyme glutamate cysteine ligase (GCL). Here, we demonstrate that TGFbeta1-induced apoptosis in the TAMH murine hepatocyte cell line is accompanied by both the cleavage and loss of the catalytic subunit of GCL (GCLC) and the down-regulation of GCLC gene expression resulting in a reduction in GCL activity and depletion of intracellular GSH. TGFbeta1-induced apoptosis is also accompanied by a reduction in Bcl-XL, an effect that may facilitate TGFbeta1-induced apoptosis as Bcl-XL overexpression inhibits TGFbeta1-induced caspase activation and cell death. Interestingly, Bcl-XL overexpression prevents TGFbeta1-induced cleavage of GCLC protein but not down-regulation of GCLC mRNA. Furthermore, TGFbeta1-induced down-regulation of GCLC mRNA is prevented by inhibition of histone deacetylase activity, suggesting that this is an active repression of GCLC gene transcription. These findings suggest that the suppression of GSH antioxidant defenses associated with the caspase-dependent cleavage of GCLC protein, caspase-independent suppression of GCLC gene expression, and depletion of intracellular GSH may play a role in enhancing TGFbeta1-induced oxidative stress and potentiating apoptotic cell death.

Overdose of the popular, and relatively safe, analgesic acetaminophen (N-acetyl-p-aminophenol, APAP, paracetamol) can produce a fatal centrilobular liver injury. APAP-induced cell death was investigated in a differentiated, transforming growth factor alpha (TGFalpha)-overexpressing, hepatocyte cell line and found to occur at concentrations, and over time frames, relevant to clinical overdose situations. Coordinated multiorganellar collapse was evident during APAP-induced cytotoxicity with widespread, yet selective, protein degradation events in vitro. Cellular proteasomal activity was inhibited with APAP treatment but not with the comparatively nonhepatotoxic APAP regioisomer, N-acetyl-m-aminophenol (AMAP). Low concentrations of the proteasome-directed inhibitor MG132 (N-carbobenzoxyl-Leu-Leu-Leucinal) increased chromatin condensation and cellular stress responses preferentially in AMAP-treated cultures, suggesting a contribution of the proteasome in APAP- but not AMAP-mediated cell death. APAP-specific alterations to mitochondria were observed morphologically with evidence of mitochondrial proliferation in vitro. Biochemical alterations to cellular proteolytic events were also found in vivo, including APAP- or AMAP-mediated inhibition of caspase-3 processing. These results indicate that, although retaining some attributes of apoptosis, both APAP- and AMAP-mediated cell death have additional distinctive features consistent with longer term necrosis.

Apoptotic cell death is usually accompanied by activation of a family of cysteine proteases termed caspases. Caspases mediate the selective proteolysis of multiple cellular targets often resulting in the disruption of survival pathways. Intracellular levels of the antioxidant glutathione (GSH) are an important determinant of cellular susceptibility to apoptosis. The rate-limiting step in GSH biosynthesis is mediated by glutamate-L-cysteine ligase (GCL), a heterodimeric enzyme consisting of a catalytic (GCLC) and a modifier (GCLM) subunit. In this report we demonstrate that GCLC is a direct target for caspase-mediated cleavage in multiple models of apoptotic cell death. Mutational analysis revealed that caspase-mediated cleavage of GCLC occurs at Asp(499) within the sequence AVVD(499)G. GCLC cleavage occurs upstream of Cys(553), which is thought to be important for association with GCLM. GCLC cleavage is accompanied by a rapid loss of intracellular GSH due to caspase-mediated extrusion of GSH from the cell. However, while GCLC cleavage is dependent on caspase-3, GSH extrusion occurs by a caspase-3-independent mechanism. Our identification of GCLC as a target for caspase-3-dependent cleavage during apoptotic cell death suggests that this post-translational modification may represent a novel mechanism for regulating GSH biosynthesis during apoptosis.

Tumor necrosis factor (TNF) is a mediator of the acute phase response in the liver and can initiate proliferation and cause cell death in hepatocytes. We investigated the mechanisms by which TNF causes apoptosis in hepatocytes focusing on the role of oxidative stress, antioxidant defenses, and mitochondrial damage. The studies were conducted in cultured AML12 cells, a line of differentiated murine hepatocytes. As is the case for hepatocytes in vivo, AML12 cells were not sensitive to cell death by TNF alone, but died by apoptosis when exposed to TNF and a small dose of actinomycin D (Act D). Morphological signs of apoptosis were not detected until 6 hours after the treatment and by 18 hours approximately 50% of the cells had died. Exposure of the cells to TNF+Act D did not block NFkappaB nuclear translocation, DNA binding, or its overall transactivation capacity. Induction of apoptosis was characterized by oxidative stress indicated by the loss of NAD(P)H and glutathione followed by mitochondrial damage that included loss of mitochondrial membrane potential, inner membrane structural damage, and mitochondrial condensation. These changes coincided with cytochrome C release and the activation of caspases-8, -9, and -3. TNF-induced apoptosis was dependent on glutathione levels. In cells with decreased levels of glutathione, TNF by itself in the absence of transcriptional blocking acted as an apoptotic agent. Conversely, the antioxidant alpha-lipoic acid, that protected against the loss of glutathione in cells exposed to TNF+Act D completely prevented mitochondrial damage, caspase activation, cytochrome C release, and apoptosis. The results demonstrate that apoptosis induced by TNF+Act D in AML12 cells involves oxidative injury and mitochondrial damage. As injury was regulated to a larger extent by the glutathione content of the cells, we suggest that the combination of TNF+Act D causes apoptosis because Act D blocks the transcription of genes required for antioxidant defenses.

Background: Etoposide mediates its cytotoxicity by inducing apoptosis. Thus, mechanisms which regulate apoptosis should also affect drug resistance. Oxidants and antioxidants have been shown to participate in the regulation of apoptosis. We were interested in studying whether responsiveness of acute myeloblastic leukemia (AML) cells to etoposide is mediated by oxidative stress and glutathione levels., Patients and Methods: Two subclones of the OCI/AML-2 cell line which are etoposide-sensitive (ES), and etoposide-resistant (ER), were established by the authors at the University of Oulu, and used as models. Assays for apoptosis included externalization of phosphatidylserine (as evidenced by annexin V binding), and caspase activation as indicated by cleavage of poly(ADP-ribose)polymerase (Western blotting). Peroxide formation was analyzed by flow cytometry. Glutathione and gamma-glutamylcysteine synthetase (gamma-GCS) levels were determined spectrophotometrically and by Western blotting, respectively., Results: Etoposide-induced apoptosis was evident 12 hours after treatment in the ES subclone, but was apparent in the ER subclone only after 24 hours. The basal glutathione and gamma-GCS levels were higher in the ER than the ES subclone. Etoposide increased peroxide formation in both subclones after 12-hour exposure. Significant depletion of glutathione was observed in the ES subclone during etoposide exposure, while glutathione levels were maintained in the ER subclone. In neither of the subclones was induction of gamma-GCS observed during 24-hour exposure to etoposide. Furthermore, the catalytic subunit of gamma-GCS was cleaved during apoptosis, concurrent with depletion of intracellular glutathione. When glutathione was depleted by treatment with buthionine sulfoximine, a direct inhibitor of gamma-GCS, the sensitivity to etoposide was increased, particularly in the ER subclone., Conclusions: The results underline the significance of glutathione biosynthesis in the responsiveness of AML cells to etoposide. The molecular mechanisms mediating glutathione depletion during etoposide exposure might include the cleavage of the catalytic subunit of gamma-GCS.

Recent studies have suggested that MAP kinase phosphatase 1 (MKP-1) is overexpressed in prostate cancer. To evaluate the role of MKP-1 in regulating cell death and tumor growth in prostate cancer, MKP-1 was conditionally overexpressed in the human prostate cancer cell line DU145. Overexpression of MKP-1 in DU145 cells blocked activation of stress-activated protein kinase (SAPK/JNK). MKP-1 overexpression in DU-145 cells was also found to inhibit Fas ligand (FasL)-induced apoptosis, as well as block the activation of caspases by Fas engagement. In addition, MKP-1 blocked the activation of apoptosis by transfected MEKK-1 and ASK-1, presumably through its inhibition of the SAPK/JNK family of enzymes. MKP-1 blocked the ability of FasL to induce loss of mitochondrial transmembrane potential (delta Psi(m)), suggesting that MKP-1 acts upstream of mitochondrial pro-apoptotic events induced by FasL and that the SAPK/JNK pathway may form the signaling link between Fas receptor and mitochondrial dysfunction. Thus, MKP-1 overexpression in prostate cancer may play a role in promoting prostate carcinogenesis by inhibiting FasL-induced cell death.