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While the earliest published health disparity research in the United States dates to 1899, the field was not formally established until the late 20th century. Initially focused on race and ethnicity, the field has broadened to include socioeconomic status. Several measures have been developed to quantify socioeconomic disadvantage, including the Social Vulnerability Index, Area Deprivation Index, and Child Opportunity Index. These indices have been validated and demonstrate correlation with health outcomes. However, socioeconomic status cannot fully explain health inequities experienced by people of minoritized racial and ethnic identities. Three generations of health disparities research have been described-identification of disparities, root analysis, and development of interventions to mitigate health inequities. While there has been an increase in publication of health disparity research, there is little third generation work. It is imperative that health disparities research move beyond defining the problem and toward interventions that will reduce health inequities. LEVELS OF EVIDENCE: Level IV., Competing Interests: Conflicts of interest We have no competing interests to declare., (Copyright © 2024 Elsevier Inc. All rights reserved.)

As the transgender population in the United States grows, gender-affirming care is becoming increasingly relevant to the practice of pediatric surgery. Medical care for the transgender and gender diverse population is a politically charged topic with significant complexity and opportunities for clarification. It is important for providers to better understand this population's unique health and social needs. This review aims to debunk long-standing myths regarding gender-affirming care and highlight the current therapeutic and legislative landscapes within the scope of pediatric surgical practice. LEVEL OF EVIDENCE: IV., Competing Interests: Conflicts of interest We have no competing interests to declare., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Here we present the 3-year results of ZUMA-4, a phase I/II multicenter study evaluating the safety and efficacy of KTEX19, an autologous anti-CD19 chimeric antigen receptor (CAR) T-cell therapy, in pediatric/adolescent patients with relapsed/refractory B-cell acute lymphoblastic leukemia. Phase I explored two dose levels and formulations. The primary endpoint was the incidence of dose-limiting toxicities. Thirty-one patients were enrolled; KTE-X19 was administered to 24 patients (median age 13.5 years, range 3-20; median follow-up 36.1 months). No dose-limiting toxicities were observed. All treated patients had grade ≥3 adverse events, commonly hypotension (50%) and anemia (42%). Grade 3 cytokine release syndrome rates were 33% in all treated patients, 75% in patients given the dose of 2×106 CAR T cells/kg, 27% in patients given the dose of 1×106 cells/kg in the 68 mL formulation, and 22% in patients given the dose of 1×106 cells/kg in the 40 mL formulation; the percentages of patients experiencing grade ≥3 neurologic events were 21%, 25%, 27%, and 11% respectively. Overall complete remission rates (including complete remission with incomplete hematologic recovery) were 67% in all treated patients, 75% in patients given 2×106 CAR T cells/kg, 64% in patients given 1×106 cells/kg in the 68 mL formulation, and 67% in patients given 1×106 cells/kg in the 40 mL formulation. Overall minimal residual diseasenegativity rates were 100% among responders; 88% of responders underwent subsequent allogeneic stem-cell transplantation. In the 1×106 (40 mL) group (recommended phase II dose), the median duration of remission censored at allogeneic stem-cell transplantation and median overall survival were not reached. Pediatric/adolescent patients with relapsed/refractory B-cell acute lymphoblastic leukemia achieved high minimal residual disease-negative remission rates with a manageable safety profile after a single dose of KTE-X19. Phase II of the study is ongoing at the dose of 1×106 CAR T cells/kg in the 40 mL formulation. ClinicalTrials.gov: NCT02625480.

Minimally invasive surgery has moved from the fringe of pediatric surgery to the mainstream to address a variety of problems. Pancreatic pathology, though uncommon and complex, is frequently amenable to laparoscopic intervention. Indications for pediatric pancreatic operative intervention includes trauma, congenital hyperinsulinemia and neoplasm. Children may require distal pancreatectomy, subtotal pancreatectomy, enucleation, lateral pancreaticojejunostomy and pancreaticoduodenectomy. Of these operations, all but pancreaticoduodenectomy have been successfully described in children using a minimally invasive approach. Traumatic transection of the main pancreatic duct may require operative intervention if endoscopic techniques are unsuccessful. Distal pancreatectomy has been successfully utilized in this circumstance. Additionally, near total pancreatectomy may also be performed laparoscopically although successful reports are limited. Enucleation, especially with the use of intraoperative ultrasound may avoid a large laparotomy for isolated benign masses. Finally, chronic pancreatitis resulting in a dilated main pancreatic duct may benefit from a lateral pancreaticojejunostomy. This operation has also successfully been performed in children. Included is a review of pediatric pancreatic minimally invasive operations paired with corresponding pathology., Competing Interests: Conflicts of Interest: Both authors have completed the ICMJE uniform disclosure form (available at http://dx.doi.org/10.21037/tgh-20-220). The series “Current Topics in Pediatric General Surgery” was commissioned by the editorial office without any funding or sponsorship. Dr. MSC served as the unpaid Guest Editor of the series. Dr. MSC reports personal fees from Bolder Surgical, outside the submitted work. The authors have no other conflicts of interest to declare., (2021 Translational Gastroenterology and Hepatology. All rights reserved.)

Competing Interests: Financial Disclosure The authors did not report any potential conflicts of interest.

Activating variants of the MAPK pathway have been found in some Langerhans cell histiocytosis (LCH) lesions. Inhibition of the MAPK pathway with trametinib (MEK inhibitor) has been shown to induce responses in LCH patients. Two adolescent males with LCH driven by BRAF p.N486_P490del have received trametinib for >1 year with no reactivation in one and partial response in another (including stable lung disease). A third male with neonatal LCH and MAP2K1p.K57_G61del had a complete response to trametinib with no active disease after 22 months. All patients continue on trametinib monotherapy with tolerable skin and creatine phosphokinase toxicity., (© 2020 Wiley Periodicals LLC.)

Purpose: We sought to compare the presentation, management, and outcomes in gastric adenocarcinoma cancer for pediatric and adult patients., Methods: Using the 2004 to 2014 National Cancer Database (NCDB), patients ≤21 years (pediatric) were retrospectively compared to >21 years (adult). Chi-squared tests were used to compare categorical variables, and Cox regression was used to estimate hazard ratios (HR) for survival differences., Results: Of the 129,024 gastric adenocarcinoma cases identified, 129 (0.10%) occurred in pediatric patients. Pediatric cases presented with more advanced disease, including poorly differentiated tumors (81% vs 65%, p = 0.006) and stage 4 disease (56% vs 41%, p = 0.002). Signet ring adenocarcinoma comprised 45% of cases in the pediatric group as compared to 20% of cases in the adults (P < 0.001). Similar proportions in both groups underwent surgery. However, near-total gastrectomy was more common in the pediatric group (16% vs 6%, p < 0.001). The proportions of patients with negative margins, nodal examination, and presence of positive nodes were similar. There was no overall survival difference between the two age groups (HR 0.92, 95% Confidence interval 0.73-1.15)., Conclusion: While gastric adenocarcinoma in pediatric patients present with a more advanced stage and poorly differentiated tumors compared to adults, survival appears to be comparable., Type of Study: Retrospective cohort study., Level of Evidence: III., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: There is a movement to ensure that pediatric patients are treated in appropriately resourced hospitals through the ACS Children's Surgery Verification (CSV) program. The objective of this study was to assess the potential difference in care provision, health outcomes and healthcare and societal costs after implementation of the CSV program., Methods: All 2011 inpatient admissions for selected complex pediatric patients warranting treatment at a hospital with Level I resources were evaluated across 6 states. Multivariate regressions were used to analyze differences in healthcare outcomes (postoperative complications including death, length of stay, readmissions and ED visits within 30 days) and costs by CSV level. Recycled predictions were used to estimate differences between the base case scenario, where children actually received care, and the optimized scenario, where all children were theoretically treated at Level I centers., Results: 8,006 children (mean age 3.06 years, SD 4.49) met inclusion criteria, with 45% treated at Level I hospitals, 30% at Level II and 25% at Level III. No statistically significant differences were observed in healthcare outcomes. Readmissions within 30 days were higher at Level II compared to Level I centers (adjusted IRR 1.61; 95% CI 1.11, 2.34), with an estimated 24 avoidable readmissions per 1000 children if treatment were shifted from Level II to Level I centers. Overall, costs per child were not significantly different between the base case and the optimized scenario., Conclusion: Many complex surgical procedures are being performed at Level II/III centers. This study found no statistically significant increase in healthcare or societal costs if these were performed instead at Level I centers under the optimized scenario. Ongoing evaluation of efforts to match institutional resources with individual patient needs is needed to optimize children's surgical care in the United States., Level of Evidence: II., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Standard of care for unilateral nephroblastoma includes total nephrectomy (TN) with nodal sampling. We sought to compare the outcomes of TN and partial nephrectomy (PN). We performed a retrospective cohort study of TN and PN for nephroblastoma using the National Cancer Data Base. The outcomes included nodal sampling frequency, margin status, and survival. Categorical and continuous data were evaluated with χ2 and t tests, respectively (P < 0.05). Generalized linear models evaluated nodal sampling and margin status. Cox regression compared survival. In total, 235 patients underwent PN and 3572 had TN. TN patients were 50 per cent more likely to undergo nodal sampling (RR: 1.47, 95% CI 1.30-1.66). There was no difference in margin status (RR: 0.91, 95% CI 0.65-1.28) or overall survival (HR 1.57; 95% CI 0.78-3.19). This study reports the largest review of patients with PN for unilateral nephroblastoma. PN patients had less nodal sampling but similar margin involvement and overall survival.

Purpose: Pediatric breast malignancies are rare, and descriptions in the literature are limited. The purpose of our study was to compare pediatric and adult breast malignancy., Methods: We performed a retrospective cohort study using the National Cancer Data Base comparing patients ≤21 years to those >21 years at diagnosis (1998-2012). Generalized linear models estimated differences in demographic, tumor, and treatment characteristics. Cox regression was used to compare overall survival., Results: Of 1,999,181 cases of invasive breast malignancies, 477 (0.02%) occurred in patients ≤21 years. Ninety-nine percent of adult patients had invasive carcinoma compared with 64.8% of pediatric patients with the remaining patients having sarcoma, malignant phyllodes, or malignancy not otherwise specified (p < 0.001). Pediatric patients were twice as likely to have an undifferentiated malignancy [relative risk (RR) 2.19; 95% confidence interval (CI) 1.72-3.79]. Half of adults presented with Stage I disease compared with only 22.7% of pediatric patients (p < 0.001). Pediatric patients were 40% more likely to have positive axillary nodes (RR 1.42; 95% CI 1.10-1.84). Among patients with invasive carcinoma, pediatric patients were more than four times as likely to receive a bilateral than a unilateral mastectomy compared with adults (RR 4.56; 95% CI 3.19-6.53). There was no difference in overall survival between children and adults., Conclusions: Pediatric breast malignancies are more advanced at presentation, and there is variability in treatment practices. Adult and pediatric patients with invasive carcinoma have similar overall survival.

Study Objective: To describe secular trends in operative experience for surgical trainees across an extended period using the most comprehensive data available, the Accreditation Council for Graduate Medical Education (ACGME) case logs., Background: Some experts have expressed concern that current trainees are inadequately prepared for independent practice. One frequently mentioned factor is whether duty hours' restrictions (DHR) implemented in 2003 and 2004 contributed by reducing time spent in the operating room., Methods: A dataset was generated from annual ACGME reports. Operative volume for total major cases (TMC), defined categories, and four index laparoscopic procedures was evaluated., Results: TMC dropped after implementation of DHR but rebounded after a transition period (949 vs 946 cases, P = nonsignificance). Abdominal cases increased from 22% of overall cases to 31%. Alimentary cases increased from 21% to 26%. Trauma and vascular surgery substantially decreased. For trauma, this drop took place well before DHR. The decrease in vascular surgery also began before DHR but continued afterward as well: 148 cases/resident in the late 1990s to 107 currently., Conclusions: Although total operative volume rebounded after implementation of DHR, diversity of operative experienced narrowed. The combined increase in alimentary and abdominal cases is nearly 13%, over a half-year's worth of operating in 5-year training programs. Bedrock general surgery cases-trauma, vascular, pediatrics, and breast-decreased. Laparoscopic operations have steadily increased. If the competence of current graduates has, in fact, diminished. Our analysis suggests that operative volume is not the problem. Rather, changing disease processes, subspecialization, reductions in resident autonomy, and technical innovation challenge how today's general surgeons are trained.

Background: Despite six randomized trials of various treatments for pediatric para-pneumonic effusion (PPE), management approaches differ. The purpose of this study was to gain insight into opinions on PPE treatment with the goal of designing a definitive trial to generate consensus intervention guidelines., Methods: To evaluate physician opinions regarding PPE management, we developed a survey based on input from a nationwide, multi-disciplinary advisory group that established content validity. The survey was disseminated broadly to six pediatric medicine and interventional radiology groups. Descriptive and χ 2 statistics were calculated., Results: There were 741 respondents (response rate 13.1%), of whom 52.2% were surgeons, 15.2% hospitalists, 14.2% pulmonologists, 12.4% intensivists, and 6.0% interventional radiologists. Nearly all respondents (97.3%) reported caring primarily for pediatric patients. Eighty percent reported no written institutional treatment guidelines. Nearly all (90.3%) agreed that patients require antibiotics, but there was disagreement regarding their duration. Respondents also were split as to how often PPE required drainage. There were multiple absolute indications for drainage, including mediastinal shift on chest radiograph (67.2%) and loculations on imaging (47.7%). There were substantial differences in the preferred first-line methods of drainage based on the treating physician's specialty, with surgeons preferring tube thoracostomy and a fibrinolytic agent (42.0%) or video-assisted thoracoscopic surgery (41.6%), whereas interventional radiologists preferred either a tube thoracostomy (46.4%) or a tube thoracostomy with a fibrinolytic agent (39.3%) (p < 0.001). A large majority (75.3%) believed that the published evidence does not identify the optimal intervention., Conclusions: There is a lack of consensus regarding the optimal treatment of PPE. Respondents believed the published evidence is inconclusive and were willing to participate in a prospective trial. These findings will help inform the design of a randomized, pragmatic clinical trial to optimize PPE management.

Background: Though the volume-outcome relationship has been well-established in adults, low mortality rates and small sample sizes have precluded definitive demonstration in children. This study compares treatment-specific factors for children with nephroblastoma at high (HVC) versus low volume centers (LVC)., Methods: We performed a retrospective cohort study comparing patients ≤18years with unilateral nephroblastoma treated at HVCs and LVCs using the National Cancer Data Base (1998-2012). Definitions of HVCs included performing above the median, the upper two quartiles, and the highest decile of nephroblastoma resections. Outcomes included nodal sampling, margin status, time to chemotherapy and radiation, and survival. Statistical analyses included χ 2 , t-tests, generalized linear, and Cox regression models (p<0.05)., Results: Of 2911 patients from 210 centers, 1443 (49.6%) were treated at HVCs. There was no difference in frequency of preoperative biopsy or days to radiation (p>0.05). High volume centers were more likely to perform nodal sampling (RR 1.04, 95%CI 1.01-1.08) and had fewer days to chemotherapy (RR 0.80, 95%CI 0.69-0.93). Five-year survival was similar (HVC: 0.93, 95%CI 0.92-0.94; LVC: 0.93, 95%CI 0.91-0.94)., Conclusions: HVCs were more likely to perform nodal sampling and had fewer days to chemotherapy. There was no difference in days to radiation or survival between centers., Level of Evidence: Level II (retrospective prognosis study)., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Importance: Melanoma in children is rare, accounting for approximately 2% of all pediatric malignant neoplasms. However, for the past 30 years, the incidence of melanoma in those younger than 20 years has been increasing. Location of the primary tumor has been shown to be an important prognostic factor, with melanomas of the scalp and neck conferring a worse prognosis than those originating at other sites., Objective: To examine the survival, demographic, tumor, and treatment characteristics of pediatric head and neck melanoma., Design, Setting, and Participants: We performed a retrospective cohort study using information from the National Cancer Data Base from January 1, 1998, to December 31, 2012, on pediatric (≤18 years) and adult (>18 years) patients with head and neck melanoma. Data analysis was conducted from August 1, 2015, to June 30, 2016., Exposure: Pediatric age (≤18 years) at diagnosis of head and neck melanoma., Main Outcomes and Measures: Survival differences were estimated using a Cox proportional hazards regression model. Surgical outcomes, including nodal sampling and margin status, were estimated with generalized linear models comparing pediatric and adult patients. Patient demographic, tumor, and treatment characteristics were estimated using t tests and χ2 tests between pediatric and adult patients with head and neck melanoma for continuous and categorical data, respectively., Results: Of the 84 744 patients with head and neck melanoma, 657 (0.8%) were 18 years or younger (mean [SD] age, 13.5 [4.7] years; 285 female and 372 male; 610 white). Pediatric and adult patients had similar demographics but different histologic subtypes (risk difference of pediatric vs adult patients: melanoma, not otherwise specified, 8.5% [95% CI, 4.7%-12.3%]; superficial spreading, 4.2% [95% CI, 0.89%-7.4%]; and lentigo maligna, -13.4% [95% CI, -14.1% to 12.6%]). Pediatric patients had tumors of similar mean depth to those in adult patients (pediatric, 1.54 mm; adult; 1.39 mm; absolute difference, 0.15 mm; [95% CI, -0.32 to 0.008]) and more frequent nodal metastases than did adult patients (risk difference of pediatric vs adult patients for stage T2, 23.9% [95% CI, 14.1%-33.6%]). Five-year survival among pediatric patients was higher for those with stage 1, 2, or 3 disease (absolute difference of pediatric vs adult patients: stage 1, 18% [95% CI, 9.7%-26.3%]; stage 2, 36% [95% CI, 25.3%-46.7%]; stage 3, 39% [95% CI, 26.8%-51.2%]; and stage 4, 2% [95% CI, -8.2% to 12.2%])., Conclusions and Relevance: Although pediatric patients with head and neck melanoma present with similar tumor depth and more frequent nodal metastases than do adult patients, younger patients have higher overall survival.

Robotic technology is being utilized in multiple hepatobiliary procedures, including hepatic resections. The benefits of minimally invasive surgical approaches have been well documented; however, there is some concern that robotic liver surgery may be prohibitively costly and therefore should be limited on this basis. A single-institution, retrospective cohort study was performed of robotic and open liver resections performed for benign and malignant pathologies. Clinical and cost outcomes were analyzed using adjusted generalized linear regression models. Clinical and cost data for 71 robotic (RH) and 88 open (OH) hepatectomies were analyzed. Operative time was significantly longer in the RH group (303 vs. 253 min; p = 0.004). Length of stay was more than 2 days shorter in the RH group (4.2 vs. 6.5 days; p < 0.001). RH perioperative costs were higher ($6026 vs. $5479; p = 0.047); however, postoperative costs were significantly lower, resulting in lower total hospital direct costs compared with OH controls ($14,754 vs. $18,998; p = 0.001). Robotic assistance is safe and effective while performing major and minor liver resections. Despite increased perioperative costs, overall RH direct costs are not greater than OH, the current standard of care.

Background/purpose: Children requiring gastrostomy/gastrojejunostomy tubes (GT/GJ) are heterogeneous and medically complex patients with high resource utilization. We created and implemented a hospital-wide standardized pathway for feeding device placement. This study compares hospital resource utilization before and after pathway implementation., Methods: We performed a retrospective cohort study comparing outcomes through one year of follow-up for consecutive groups of children undergoing GT/GJ placement prepathway (n=298, 1/1/2010-12/31/2011) and postpathway (n=140, 6/1/2013-7/31/2014) implementation. We determined the change in the rate of hospital resource utilization events and time to first event., Results: Prior to implementation, 145 (48.7%) devices were placed surgically, 113 (37.9%) endoscopically and 40 (13.4%) using image guidance. After implementation, 102 (72.9%) were placed surgically, 23 (16.4%) endoscopically and 15 (10.7%) using image guidance. Prior to implementation, 174/298 (58.4%) patients required additional hospital resource utilization compared to 60/143 (42.0%) corresponding to a multivariate adjusted 38% reduced risk of a subsequent feeding tube related event., Conclusions: Care of tube-feeding dependent patients is spread among multiple specialists leading to variability in the preoperative workup, intraoperative technique and postoperative care. Our study shows an association between implementation of a standardized pathway and a decrease in hospital resource utilization., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background: Unplanned readmissions are costly to family satisfaction and negatively associated with quality of care. We hypothesized that patient, operative, and hospital factors would be associated with pediatric readmission., Methods: All patients with an inpatient operation from 10/1/2008 to 7/28/2014 at a freestanding children's hospital were included. A retrospective cohort study using multivariable forward stepwise logistic regression determined factors associated with unplanned readmission within 30 days of discharge., Results: Among 20,785 patients with an operation there were 26,978 encounters and 3,092 readmissions (11.5%). Thirteen of 33 candidate variables considered in the stepwise regression were significantly associated with readmission. Patients with an emergency department visit within 365 days of operation, American Society of Anesthesiologists class 4 or greater, Hispanic ethnicity and late-day or holiday/weekend discharges were more likely to have an unplanned readmission (odds ratio [OR] = 1.96; 95% confidence interval [CI] = 1.76 to 2.19, OR = 2.00; 95% CI = 1.58 to 2.53, OR = 1.16; 95% CI = 1.04 to 1.29, OR = 2.27; 95% CI = 1.55 to 3.63. respectively)., Conclusions: Patient and hospital factors may be associated with readmission. Day and time of discharge represent variability of care and are important targets for hospital initiatives to decrease unplanned readmission., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Background/purpose: Optimal outcomes for necrotizing soft tissue infections (NSTI) depend on rapid diagnosis and management. The Laboratory Risk Indicator for Necrotizing Fasciitis (LRINEC) score is a validated diagnostic tool for adult NSTI, but its value for children remains unknown. We hypothesized that modification of the LRINEC score may increase its diagnostic accuracy for pediatric NSTI., Methods: We performed a case-control study of pediatric patients (age <18) with NSTI (cases) and patients with severe soft tissue infections prompting surgical consultation (controls). The LRINEC score was calculated for cases and controls and compared to a modified, pediatric LRINEC (P-LRINEC) score. Diagnostic accuracy was analyzed through receiver operating characteristic (ROC) curves., Results: From 2010 to 2014, 20 cases and 20 controls were identified at two children's hospitals. Median LRINEC score was 3.5 (1-8) for cases and 2 (1-7) for controls (p=0.03). The P-LRINEC was comprised of serum CRP >20 (sensitivity=95% (95%CI 79-100%)) and serum sodium <135 (specificity=95% (95%CI 82-100%)). Area under ROC curves was 0.70 (95%CI 0.54-0.87) for the LRINEC score and 0.84 (95%CI 0.72-0.96) for the P-LRINEC score (p=0.06)., Conclusion: The P-LRINEC is a simplified version of the LRINEC score utilizing only CRP and sodium and may provide superior accuracy in predicting pediatric NSTI., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Objective: To assess associations between primary cesarean delivery and adverse delivery outcomes with very advanced maternal age., Study Design: We conducted a population-based cohort study including 78,880 births to mothers 25 years and older with singleton births from 2003 to 2012 using Washington State birth certificates and hospital discharge data, excluding births to women with a prior cesarean section. The primary outcome was mode of delivery. Secondary outcomes included maternal transfusion, chorioamnionitis, severe perineal lacerations and prolonged length of stay. Outcomes of births to women of advanced maternal age (35 to 39, 40 to 44) and very advanced maternal age (45 to 49, ⩾50) were compared with referent births among women aged 25 to 34 years. General linear models with a log-link function were used to calculate unadjusted and adjusted relative risks and 95% confidence intervals (CIs)., Result: Proportions and risks of primary cesarean section increased with age (25 to 34 years, referent: 20.0%; 35 to 39 years: 25.9%, relative risk (RR)=1.25 (95% CI=1.20 to 1.29); 40 to 44 years: 30.9%, RR=1.45 (95% CI=1.40 to 1.50); 45 to 49 years: 35.7%, RR=1.59 (95% CI=1.45 to 1.75); and ⩾50 years: 60.7%, RR=2.44 (95% CI=1.95 to 3.05); P-trend <0.001). Associations did not differ between primiparous and multiparous women. No differences were noted for measures of maternal morbidity, except there was a trend of increasing risk of prolonged length of stay among births to older women (P-trend <0.001)., Conclusion: Primary cesarean delivery risk continues to increase above age 35 regardless of prior vaginal birth, with the highest risk among women aged 50 years and older.

Importance: Nasopharyngeal carcinoma (NPC) is endemic in some Asian regions but is uncommon in the United States. Little is known about the racial, demographic, and biological characteristics of the disease in pediatric patients., Objectives: To improve understanding of the differences between pediatric and adult NPC and to determine whether race conferred a survival difference among pediatric patients with NPC., Design, Setting, and Participants: This retrospective cohort study included all 17 317 patients with a primary diagnosis of NCP in the National Cancer Data Base from January 1, 1998, to December 31, 2011. Of these, 699 patients were 21 years or younger (pediatric); 16 618 patients, older than 21 years (adult). Data were analyzed after data collection., Exposure: Pediatric age at diagnosis of NPC., Main Outcomes and Measures: Demographic, tumor, and treatment characteristics of pediatric patients with NPC were compared with those of adults using the χ2 test for categorical variables. An adjusted Cox proportional hazards regression model was used to examine survival differences in pediatric patients relative to adult patients. In addition, the risk for pediatric mortality by race was estimated., Results: Of the 17 317 patients, a total of 699 pediatric and 16 618 adult patients were identified with a primary diagnosis of NPC (female, 239 pediatric patients [34.2%] and 5153 adult patients [32.4%]). Pediatric patients were most commonly black (299 of 686 [43.6%]), whereas adults were most likely to be non-Hispanic white (9839 of 16 504 [60.0%]; P < .001). Pediatric patients were less likely to be Asian (39 of 686 [5.7%]) than were adults (3226 of 16 405 [19.7%]; P < .001). Pediatric patients were more likely to have regional nodal evaluation and to present with stage IV disease (227 of 643 [35.3%] and 330 of 565 [58.4%], respectively) than were adult patients (3748 of 15 631 [24.0%] and 6553 of 13 721 [47.8%], respectively; P < .001 for both comparisons). Pediatric patients had a lower risk for mortality relative to adults (hazard ratio, 0.37; 95% CI, 0.25-0.56). No difference in mortality by racial group was found among pediatric patients (hazard ratio, 1.10; 95% CI, 0.82-1.40)., Conclusions and Relevance: Pediatric patients with NPC were more commonly black and presented more frequently with stage IV disease. Pediatric patients had a decreased mortality risk relative to adults, even after adjusting for covariables. Asian race was not associated with increased mortality in pediatric patients with NPC. Racial differences are not associated with an increased risk for mortality among pediatric patients.

Patient-centered care is an expectation in our current environment, yet it is only one of the six domains that the Institute of Medicine has described as critical in redesigning the architecture of a medical system. Patients requiring long-term feeding tube access represent a particularly complex group of patients who stress the mechanisms placed within a healthcare system to optimize quality and safety. We describe the implementation of a new approach to this patient population that serves as an example of redesigning a system of care to optimize safety using the principles of patient-centered care while delivering safe, effective, timely, efficient, and equitable care., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: The effects of hospital location and designation on postoperative pediatric outcomes remain unclear. We hypothesized that urban hospital outcomes would be superior to rural hospitals, and that outcomes at urban centers would differ for children from rural versus urban counties., Methods: Retrospective cohort study of children undergoing appendectomy (n=129,507) and pyloromyotomy (n=13,452) using the 2006/2009 KID databases. Hospitals were characterized by specialty designation and classified as urban/rural. County of residence was classified as urban/rural. Outcomes included complications and length of stay. Multivariate regression models were used to adjust for confounding., Results: Among appendectomy patients, treatment at urban hospitals was associated with reduced odds of any postoperative complication (OR=0.77, 95% C.I. 0.70-0.85) and anesthesia-related complications (OR=0.72, 95% C.I. 0.57-0.91). This association was strongest in the youngest children (<5 years) and at children's hospitals. For pyloromyotomy patients, urban hospitals were associated with reduced odds of any complication (OR=0.43, 95% C.I. 0.24-0.75), anesthesia-related complications (OR=0.14, 95% C.I. 0.05-0.37), and duodenal perforation (OR=0.46, 95% C.I. 0.19-1.07). These associations were most significant at children's hospitals., Conclusions: Postoperative outcomes appear to be improved at urban specialty hospitals relative to rural hospitals for certain common pediatric procedures. Identification of the factors driving this association may help inform resource optimization efforts in pediatric surgery., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: Safety concerns about the use of radiation-based imaging such as computed tomography (CT) in children have resulted in national recommendations to use ultrasound (US) for the diagnosis of appendicitis when possible. We evaluated the trends in CT and US use in a statewide sample and the accuracy of these modalities., Methods: Patients less than or equal to 18 years undergoing appendectomy in Washington State from 2008 to 2013 were evaluated for preoperative US/CT use, as well as imaging/pathology concordance using data from the Surgical Care and Outcomes Assessment Program., Results: Among 3,353 children, 98.3% underwent preoperative imaging. There was a significant increase in the use of US first over the study period (P < .001). The use of CT at any time during the evaluation decreased. Despite this, in 2013, over 40% of the children still underwent CT imaging. Concordance between US imaging and pathology varied between 40% and 75% at hospitals performing greater than or equal to 10 appendectomies in 2013. Over one third (34.9%) of CT scans performed in the evaluation of children with appendicitis were performed after an indeterminate US., Conclusions: Although the use of US as the first imaging modality to diagnose pediatric appendicitis has increased over the past 5 years, over 40% of children still undergo a CT scan during their preoperative evaluation. Causality for this persistence of CT use is unclear, but could include variability in US accuracy, lack of training, and lack of awareness of the risks of radiation-based imaging. Developing a campaign to focus on continued reduction in CT and increased use of high-quality US should be pursued., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: There are safety concerns about the use of radiation-based imaging (computed tomography [CT]) to diagnose appendicitis in children. Factors associated with CT use remain to be determined., Methods: For patients ≤18 years old undergoing appendectomy, we evaluated diagnostic imaging performed, patient characteristics, hospital type, and imaging/pathology concordance (2008-2012) using data from Washington State's Surgical Care and Outcomes Assessment Program., Results: Among 2538 children, 99.7% underwent pre-operative imaging. 52.7% had a CT scan as their first study. After adjustment, age >10 years (OR 2.9 (95% CI 2.2-4.0), Hispanic ethnicity (OR 1.7, 95% CI 1.5-1.9), and being obese (OR 1.7, 95% CI 1.4-2.1) were associated with CT use first. Evaluation at a non-children's hospital was associated with higher odds of CT use (OR 7.9, 95% CI 7.5-8.4). Ultrasound concordance with pathology was higher for males (72.3 vs. 66.4%, p=.03), in perforated appendicitis (75.9 vs. 67.5%, p=.009), and at children's hospitals compared to general adult hospitals (77.3 vs. 62.2%, p<.001). CT use has decreased yearly statewide., Conclusions: Over 50% of children with appendicitis had radiation-based imaging. Understanding factors associated with CT use should allow for more specific QI interventions to reduce radiation exposure. Site of care remains a significant factor in radiation exposure for children., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Importance: Minimally invasive surgery (MIS) has created a shift in how many surgical diseases are treated. Examining the effect on resident operative experience provides valuable insight into trends that may be useful for restructuring the requirements of resident training., Objective: To evaluate changes in general surgery resident operative experience regarding MIS., Design, Setting, and Participants: Retrospective review of the frequency of MIS relative to open operations among general surgery residents using the Accreditation Council for Graduate Medical Education case logs for academic years 1993-1994 through 2011-2012., Exposures: General surgery residency training among accredited programs in the United States., Main Outcomes and Measures: We analyzed the difference in the mean number of MIS techniques and corresponding open procedures across training periods using 2-tailed t tests with statistical significance set at P < .05., Results: Of 6,467,708 operations with the option of MIS, 2,393,030 (37.0%) were performed with the MIS approach. Of all MIS operations performed, the 5 most common were cholecystectomy (48.5%), appendectomy (16.2%), groin hernia repair (10.0%), abdominal exploration (nontrauma) (4.4%), and antireflux procedures (3.6%). During the study period, there was a transition from a predominantly open to MIS approach for appendectomy, antireflux procedures, thoracic wedge resection, and partial gastric resection. Cholecystectomy is the only procedure for which MIS was more common than the open technique throughout the study period (P < .001). The open approach is more common for all other procedures, including splenectomy (0.7% MIS), common bile duct exploration (24.9% MIS), gastrostomy (25.9% MIS), abdominal exploration (33.1% MIS), hernia (20.3% MIS), lung resection (22.3% MIS), partial or total colectomy (39.1%), enterolysis (19.0% MIS), ileostomy (9.0% MIS), enterectomy (5.2% MIS), vagotomy (1.8% MIS), and pediatric antireflux procedures (35.9% MIS); P < .001., Conclusions and Relevance: Minimally invasive surgery has an increasingly prominent role in contemporary surgical therapy for many common diseases. The open approach, however, still predominates in all but 5 procedures. Residents today must become efficient at performing multiple techniques for a single procedure, which demands a broader skill set than in the past.

Background: Minimally invasive parathyroidectomy (MIP) is dependent upon accurate preoperative parathyroid localization. We hypothesized that surgeon recognition of subtle differences in radiotracer accumulation would increase the sensitivity of technetium-99m sestamibi imaging and result in more frequent use of MIP., Methods: Technetium-99m sestamibi scans completed at our institution for patients who underwent resection of a solitary parathyroid adenoma were reviewed by a surgeon and a radiologist who were blinded to patient identifying information, prior scan interpretation, and results of the operation. For each scan, the reviewer determined whether there was abnormal radiotracer accumulation and documented its location. Results were correlated with outcome of operation and final pathology. Blinded interpretations of the surgeon and radiologist were compared to each other and to the original radiologic interpretation., Results: From 1994 to 2009, 274 patients with primary hyperparathyroidism (HPT) had sestamibi imaging prior to parathyroidectomy; 149 patients with a single adenoma underwent curative parathyroidectomy and had scans available for review. Seventeen radiologists who reviewed an average of 11 ± 14 scans (range = 1-61) completed the original interpretations of the sestamibi imaging. Sensitivity of sestamibi imaging was 86% for the blinded surgeon compared to 75% for the blinded radiologist and 69% for the original radiologists (P < 0.05). There was no difference in the false positive rates (blinded surgeon = 5%, blinded radiologist = 5%, original radiologists = 5%, P > 0.05)., Conclusion: Radiologists were less likely to call a scan positive. Surgeon recognition of subtle anatomic asymmetry increases the sensitivity of sestamibi imaging and successful completion of MIP., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Limited information is available regarding the use of amifostine in pediatric hematopoietic stem cell transplant (HSCT) patients. Melphalan, carboplatin, etoposide +/- cyclophosphamide is a commonly used preparatory regimen in pediatric solid tumor HSCT. Therefore, we decided to determine the feasibility of the addition of amifostine (750 mg/m b.i.d. x 4 d) to melphalan (200 mg/m), carboplatin (1200 mg/m), and etoposide (800 mg/m) (level 1) and escalating doses of cyclophosphamide (3000 mg/m and 3800 mg/m, levels 2 and 3, respectively) followed by autologous HSCT. Thirty-two patients with a variety of pediatric solid tumors were studied. Seventeen patients were accrued at level 1, 9 at level 2, and 6 at level 3. Major toxicities during the administration of the preparatory regimen were hypocalcemia, emesis, and hypotension. Hypocalcemia required aggressive calcium supplementation during the conditioning phase. No dose limiting toxicities were encountered at level 3. Amifostine at 750 mg/m b.i.d. for 4 days can be administered with a double alkylator regimen consisting of melphalan (200 mg/m), cyclophosphamide (up to 3800 mg/m), carboplatin (1200 mg/m), and etoposide (800 mg/m) with manageable toxicities.

Objective: In April, May, and June 2003, we conducted market research with decision makers from 3 payer segments to determine their perspectives on the potential introduction of statins to the U.S. over-the-counter (OTC) market., Methods: We selected a convenience sample of survey participants based upon prominence in the market, membership size, and by willingness to participate in our study. The 12 participating managed care organizations (MCOs) cover approximately 100 million lives. The 4 pharmacy benefit managers (PBMs) cover approximately 200 million lives. The 3 large employers (one employer withheld quantitative results) provide medical coverage to nearly 1.4 million employees, both actives and retirees. Each survey participant received a verbal description of an OTC statin and a proposed patient self-management system. We asked each participant a series of questions to obtain opinions on a number of issues related to the potential introduction of an OTC statin., Results: Our research findings can be summarized in the following key conclusions: (1) MCO representatives generally view OTC statins as a low-risk and beneficial addition to drug therapy options; (2) payer policies will continue to support access to prescription statins with no change in policy following introduction of an OTC option; (3) several of the MCOs and 75% of the PBMs anticipate a sharp, short-term increase in plan costs as a consequence of OTC statin availability; (4) survey participants believe that consumer or member reaction will be mixed and that consumer advertising and physician education will be important; (5) MCOs and PBMs are eager for involvement but cautious about partnerships with pharmaceutical companies; and (6) the details of any OTC statin offering will lay the foundation for its success., Conclusions: Based upon interviews conducted from April through June 2003, key decision makers from 4 PBMs, 12 MCOs, and 3 large employers generally considered the introduction of an OTC statin as a low risk and a beneficial addition to drug therapy. Most believed that increased awareness would result in an initial increase in plan costs, but long-term savings would accrue through improved care and availability of lower-cost OTC options for low-to-moderate-risk patients. The key concern is how to help patients gain enough knowledge and comfort to manage their own cholesterol therapy safely and successfully.

Granulocyte colony-stimulating factor (G-CSF) is the principal cytokine regulating granulopoiesis. G-CSF receptor-deficient mice (G-CSFR-/-) are neutropenic but have only a modest reduction of committed myeloid progenitors. Since it is likely that compensatory mechanisms are induced by the severe neutropenia present in G-CSFR-/- mice, a competitive repopulation assay was performed. These data show that under basal conditions, G-CSF drives nearly all of granulopoiesis through multiple mechanisms. Most importantly, G-CSFR signals regulate the production and/or maintenance of committed-myeloid progenitors. Surprisingly, G-CSFR signals also play a significant role in the regulation of primitive multipotential progenitors in vivo. The contribution of G-CSFR-/- cells to the hematopoietic stem cell compartment is modestly reduced. Moreover, a marked decrease in the contribution of G-CSFR-/- cells to other progenitors in the myeloid pathway, including erythroid and megakaryocytic progenitors, is observed. In contrast, relative to the hematopoietic stem cell compartment, the contribution of G-CSFR-/- cells to the lymphoid lineages is increased. These data suggest that G-CSFR signals may play a role in directing the commitment of primitive hematopoietic progenitors to the common myeloid lineage. Thus, regulation of G-CSF levels may provide a mechanism for directing primitive hematopoietic progenitors into the common myeloid lineage in response to environmental stresses.

A higher risk of obesity in stunted children has been described in Hispanic-American, Jamaican and Andean populations, but little systematic exploration has been done concerning this area in nutrition. This paper examines the relationship between stunting and overweight status for children aged 3-6 and 7-9 y in nationally representative surveys in Russia, Brazil, and the Republic of South Africa and a large nationwide survey in China. Using identical cut-offs for body mass index, the prevalence of child overweight in these countries ranges from 10.5 to 25.6% (based on the 85th percentile); recent NHANES III results indicate that this prevalence is around 22% in the U.S. Stunting is also common in the surveyed countries affecting 9.2-30.6% of all children. Our results showed a significant association between stunting and overweight status in children of all countries. The income-adjusted risk ratios of being overweight for a stunted child ranged from 1.7 to 7.8. Clearly, there is an important association between stunting and high weight-for-height in a variety of ethnic environmental and social backgrounds. Although the underlying mechanisms remain unexplored, this association has serious public health implications particularly for lower income countries. As these countries enter the nutrition transition experiencing large changes in dietary and activity patterns, they may face, among other problems, additional difficulties in their fight against obesity.

Nitric oxide synthase (NOS) catalyzes the oxidation of L-arginine to citrulline and nitric oxide. C415H and C415A mutants of the neuronal isoform of NOS (nNOS) were expressed in a baculovirus system and purified to homogeneity for spectral analysis and activity measurements. UV-visible spectra of each mutant lacked an observable Soret peak, suggesting that neither mutant contained heme. When reduced in the presence of CO, however, a small Soret centered at 417 nm could be detected for the C415H mutant, further supporting the assignment of C415 as the axial ligand to the heme. In addition to a deficiency in bound heme, neither mutant had any detectable bound tetrahydrobiopterin, as compared to wild-type enzyme, which had a ratio of 0.84 mol of bound pteridine:1 mol of nNOS 160 kDa subunit. The C415H mutant contained bound FAD and FMN at levels of 1.0 +/- 0.1 and 0.9 +/- 0.1 mol/mol of nNOS subunit, respectively. UV-visible spectra of both nNOS mutants retained the distinctive absorbance due to tightly associated oxidized flavin prosthetic groups. Further, the spectra suggested the presence of a neutral flavin semiquinone. Ferricyanide oxidation of the C415A mutant yielded a spectrum that was essentially that of oxidized flavin. Ferricyanide titration showed that the C415A mutant contained approximately 1 reducing equiv. Circular dichroism spectra suggested that each mutant was folded properly, in that both spectra were found to be essentially identical to the spectrum of wild-type nNOS. Neither mutant could synthesize nitric oxide, and neither mutant had the ability to oxidize NADPH unless an exogenous electron acceptor was added. The rate of cytochrome c reduction by each mutant was found to be slightly less, but very similar to the rate (approximately 20 mumol mg-1 min-1) observed with wild-type nNOS. In all cases, the rate of cytochrome c reduction increased approximately 15-fold with the addition of calmodulin. Overall, these spectral and activity data suggest that C415 is the axial heme ligand and that a point mutation at C415 prevents binding of heme and tetrahydrobiopterin without interfering with the global folding or the reductase function of nNOS.

Products of inducible nitric oxide synthase (iNOS) are known to be involved in lung injury following intrapulmonary deposition of immunoglobulin G immune complexes (IgG-ICx). In the current studies rat alveolar macrophages stimulated in vitro with murine interferon gamma (IFN-gamma), tumor necrosis factor alpha, interleukin 1 alpha, (IL-1 alpha), lipopolysaccharide (LPS), or IgG-ICx immunostained for iNOS and produced nitrite/nitrate- (NO2-/NO3-) in a dose- and time-dependent manner requiring availability of L-arginine. Under the same conditions, IL-4 and IL-10 reduced NO2-/NO3- generation. Type II alveolar epithelial cells, which were obtained from normal rat lungs and stimulated in vitro with IgG-ICx, LPS, or IFN-gamma, also immunostained for iNOS and generated NO2-/NO3-. Special techniques of bronchoalveolar lavage (BAL) were used to retrieve alveolar macrophages and type II alveolar epithelial cells. Under these conditions, intrapulmonary deposition of LPS yielded BAL fluids containing increased amounts of NO2-/NO3- and macrophages that spontaneously released NO2-/NO3- and stained for iNOS. After intrapulmonary deposition of IgG both macrophages as well as type II cells (retrieved by BAL) spontaneously produced NO2-/NO3- and both cell types immunostained for iNOS (approximately 20% of all type II cells and 35% of all alveolar macrophages). Using dual fluorescence staining for cell identification, frozen sections of lung tissue after IgG immune complex deposition revealed iNOS in both alveolar macrophages and type II cells. Finally, in the immune complex model of alveolitis, the appearance of iNOS in macrophages as well as macrophage production in vitro of NO2-/NO3- was dependent on the in vivo availability of tumor necrosis factor alpha, IL-1, and IFN-gamma. These studies suggest a dual cell source for nitric oxide in inflamed lungs and the requirements for iNOS of several cytokines.

Nitric oxide synthase (NOS) catalyzes the conversion of L-arginine to citrulline and nitric oxide (.NO). A baculovirus overexpression system has been developed for a constitutive NOS isoform, cloned originally from rat cerebellum (B-NOS). Recombinant virus was used at a multiplicity of infection of 5 to infect Spodoptera frugiperda cells in culture, and NOS was expressed to 10% of the total soluble protein at 48 h postinfection. In order to express catalytically active enzyme, it was necessary to supplement the culture media with hemin. This increased the activity of the enzyme 7-fold. A two column affinity purification was developed for the recombinant enzyme, which gave homogeneous protein that migrated at 150 kDa on a denaturing polyacrylamide gel. A Km for L-arginine was determined to be 2.0 +/- 0.4 microM. As isolated, recombinant B-NOS exhibited a Soret maximum at 402 nm, which shifted to 394 nm in the presence of L-arginine. The Soret maximum of the reduced enzyme in the presence of CO was 444 nm. Initial rate steady-state kinetic analysis of the recombinant B-NOS showed evidence of substrate inhibition by L-arginine, which could also be seen in a partially purified preparation of B-NOS from rat cerebella. This substrate inhibition was not observed with the inducible isoform of NOS, purified from immunostimulated murine macrophages. A C415H mutant was overexpressed and purified using the same conditions established for the wild-type recombinant B-NOS. This C415H mutant exhibited no activity and did not bind heme, providing the first experimental evidence to support previously reported primary amino acid comparisons which suggest that C415 provides the coordinating thiolate to the heme moiety in B-NOS.

.N = O synthase catalyzes the oxidation of one of the two chemically equivalent guanido nitrogens of L-arginine to nitric oxide (.N = O). NG-Methyl-L-arginine has been previously characterized as a potent competitive inhibitor of both major types of .N = O synthases. Initial rate kinetics were performed with a spectrophotometric assay based on the oxidation of oxy- to methemoglobin by .N = O. NG-Methyl-L-arginine was a competitive inhibitor of .N = O synthase activity derived from activated murine macrophages with a Ki of 6.2 microM. When the enzyme was pre-incubated in the presence of the required cofactors NADPH and tetrahydrobiopterin, time- and concentration-dependent irreversible inactivation of the activity was observed. At 37 degrees C the kinact was 0.050 min-1. This inactivation process exhibited substrate protection, saturation kinetics and required the cofactors necessary for enzymatic turnover. These data indicate that NG-methyl-L-arginine acts as a mechanism-based enzyme inactivator of murine macrophage .N = O synthase.

Avermectins with a wide range of novel C-25 substituents have been prepared by feeding carboxylic acids or their biosynthetic precursors to a Streptomyces avermitilis mutant strain ATCC 53568. This organism lacks the ability to form isobutyric and S-2-methylbutyric acids from their 2-oxo acid precursors and thus is unable to produce natural avermectins unless supplied with these acids. The novel avermectins produced by mutational biosynthesis possess broad-spectrum antiparasitic activity.

1. We describe two simple procedures for the rapid identification of certain structural features of glycolipid anchors in acetylcholinesterases (AChEs). 2. Treatment with alkaline hydroxylamine (that cleaves ester-linked acyl chains but not ether-linked alkyl chains) converts molecules possessing a diacylglycerol, but not those with an alkylacylglycerol, into hydrophilic derivatives. AChEs in human and bovine erythrocytes possess an alkylacylglycerol (Roberts et al., J. Biol. Chem. 263:18766-18775, 1988; Biochem. Biophys. Res. Commun. 150:271-277, 1988) and are not converted to hydrophilic dimers by alkaline hydroxylamine. Amphiphilic dimers of AChE from Drosophila, from mouse erythrocytes, and from the human erythroleukaemia cell line K562 also resist the treatment with hydroxylamine and likely possess a terminal alkylacylglycerol. This indicates that the cellular pool of free glycolipids used as precursors of protein anchors is distinct from the pool of membrane phosphatidylinositols (which contain diacylglycerols). 3. Pretreatment with alkaline hydroxylamine is required to render the amphiphilic AChE from human erythrocytes susceptible to digestion by Bacillus thuringiensis phosphatidylinositol-specific phospholipase C (PI-PLC) (Toutant et al., Eur. J. Biochem. 180:503-508, 1989). We show here that this is also the case for the AChE from mouse erythrocytes, which therefore likely possesses an additional acyl chain in the anchor that prevents the action of PI-PLC. 4. In two sublines of K562 cells (48 and 243), we observed that AChE either was directly susceptible to PI-PLC (243) or required a prior deacylation by alkaline hydroxylamine (48). This suggests that glycolipid anchors in AChE of K562-48 cells, but not those in AChE of K562-243 cells, contain the additional acylation demonstrated in AChE from human erythrocytes. These observations illustrate the cell specificity (and the lack of species-specificity) of the structure of glycolipid anchors.

The polyclonal antibody response of the Lewis rat to bovine myelin proteolipid protein (PLP) has been investigated following immunisation with either the purified protein or bovine central nervous system myelin. In both situations, the carboxyl-terminal sequence of PLP was identified as the immunodominant domain of this protein and epitope mapping demonstrated that the carboxyl-terminal amino acid, phenylalanine276, was a critical requirement for antibody recognition of this epitope. This single epitope accounted for approximately 78% and 56% of the antibody response to PLP in rats immunised with PLP or bovine myelin, respectively. Polyclonal rat antibodies specific for this carboxyl terminal epitope of PLP were also obtained following immunisation with a synthetic 20 amino acid oligopeptide analogue of the carboxyl-terminal sequence of PLP. Western blotting demonstrated this antibody response was specific for the PLP and DM-20 components of mammalian central nervous system myelin. In contrast, no major PLP epitopes were detected within the PLP amino acid sequences 35-58 and 91-150, the other major hydrophilic domains of this protein.

Acetylcholinesterase (AChE) in K562 cells exists in two molecular forms. The major form, an amphiphilic dimer (G2a) which sediments at 5.3 S, and the minor form, an amphiphilic monomer (G1a) which sediments at 3.5 S. Extraction in the presence of the sulfhydryl alkylating agent N-ethylmaleimide was required to preserve the G2a form. In Triton X-100 extracts of the subline K562-243, phosphatidylinositol-specific phospholipase C (PtdIns-PLC) from Bacillus thuringiensis converted most of the G2a AChE into a hydrophilic dimer (G2h), indicating that the G2a form possessed a hydrophobic glycoinositol phospholipid that mediated its attachment to the membrane. Treatment of intact K562-243 cells with PtdIns-PLC released approximately 60% of the total AChE activity and provided an estimate of the externally exposed AChE. The direct conversion from an amphiphilic to a hydrophilic dimeric form by PtdIns-PLC was not obtained in extracts or intact cells of the subline K562-48. Instead, pretreatment with alkaline hydroxylamine was necessary to render the amphiphilic G2 form of this subline susceptible to digestion by the phospholipase. In this respect, the amphiphilic dimer of K562-48 AChE resembles the G2a form of human erythrocyte AChE, which is resistant to PtdIns-PLC because of the direct palmitoylation of an inositol hydroxyl group in the anchor [Roberts et al. (1988) J. Biol. Chem. 263, 18766-18775]. Release of this acyl chain by hydroxylamine renders the enzyme susceptible to PtdIns-PLC [Toutant et al. (1989) Eur. J. Biochem. 180, 503-508]. In both K562 sublines, sialidase decreased the migration of the G2a form but not of the G1a form of AChE. G1a forms thus appear to represent an intracellular pool of newly synthesized molecules residing in a compartment proximal to the trans-Golgi apparatus. The sialidase-resistant G1a molecules were also resistant to PtdIns-PLC digestion; possible explanations for this resistance are presented.