Outcomes of Colectomy and Proctectomy According to Surgeon Training: General vs Colorectal Surgeons.

BACKGROUND: Colectomies and proctectomies are commonly performed by both general surgeons (GS) and colorectal surgeons (CRS). The aim of our study was to examine the outcomes of elective colectomy, urgent colectomy, and elective proctectomy according to surgeon training. STUDY DESIGN: Data were obtained from the Vizient database for adults who underwent elective colectomy, urgent colectomy, and elective proctectomy from 2020 to 2022. Operations performed in the setting of trauma and patients within the database's highest relative expected mortality risk group were excluded. Outcomes were compared according to surgeon's specialty: GS vs CRS. The primary outcome was in-hospital mortality. The secondary outcome was in-hospital complication rate. Data were analyzed using multivariate logistic regression. RESULTS: Of 149,516 elective colectomies, 75,711 (50.6%) were performed by GS and 73,805 (49.4%) by CRS. Compared with elective colectomies performed by CRS, elective colectomies performed by GS had higher rates of complications (4.9% vs 3.9%, odds ratio [OR] 1.23, 95% CI 1.17 to 1.29, p < 0.01) and mortality (0.5% vs 0.2%, OR 2.06, 95% CI 1.72 to 2.47, p < 0.01). Of 71,718 urgent colectomies, 54,680 (76.2%) were performed by GS, whereas 17,038 (23.8%) were performed by CRS. Compared with urgent colectomies performed by CRS, urgent colectomies performed by GS were associated with higher rates of complications (12.1% vs 10.4%, OR 1.14, 95% CI 1.08 to 1.20, p < 0.01) and mortality (5.1% vs 2.3%, OR 2.08, 95% CI 1.93 to 2.23, p < 0.01). Of 43,749 elective proctectomies, 28,458 (65.0%) were performed by CRS and 15,291 (35.0%) by GS. Compared with proctectomies performed by CRS, those performed by GS were associated with higher rates of complications (5.3% vs 4.4%, OR 1.16, 95% CI 1.06 to 1.27, p < 0.01) and mortality (0.3% vs 0.2%, OR 1.49, 95% CI 1.02 to 2.20, p = 0.04). CONCLUSIONS: In this nationwide study, colectomies and proctectomies performed by CRS were associated with improved outcomes compared with GS. Hospitals without a CRS on staff should consider prioritizing recruiting CRS specialists.

Association of Residency Applicants' Hobbies and Subsequent Technical Proficiency Ratings as General Surgery Chief Residents: A Multi-Institutional Study.

OBJECTIVE: To determine whether participation in certain hobbies (e.g., participation in sports, playing musical instruments, or other hobbies requiring fine motor skills), preresidency, are associated with higher technical skills ratings at the time of residency graduation. DESIGN: Faculty members from 14 general surgery residency programs scored individual graduates from 2017 to 2020 on their technical skills using a 5-point Likert scale. Hobbies for these residents were collected from their Electronic Residency Application Service (ERAS) data. A single reviewer classified each ERAS hobby into predefined categories including musical instruments, sports requiring hand-eye coordination, team sports, and activities necessitating hand-eye coordination. Spearman correlation coefficients were calculated for the relationship between each category of hobby-as well as the total number of hobbies in each category-and the outcome of surgical faculty ratings of residents' technical surgical skills during their last year of residency. A proportional odds model including the above predictive variables was also fit to the data. SETTING: Fourteen general surgery residency programs. PARTICIPANTS: General surgery residency graduates from 14 different programs from 2017 to 2020. RESULTS: There were 296 residents across 14 institutions. The average ranking of residents' technical skills was 3.24 (SD 1.1). A total of 40% of residents played sports involving hand-eye coordination, 31% played team sports, 28% participated in nonsport hobbies that require eye-hand coordination, and 20% played musical instruments. Correlation coefficients were not statistically significant for any of the categories. In the proportional odds model, none of the variables were associated with statistically significant increased odds of a higher technical skills rating. CONCLUSIONS: There was no correlation between general surgery chief residents' technical skills as rated by faculty, and self-reported pre-residency hobbies on the ERAS application. These findings suggest such hobbies prior to residency are unlikely to predict future technical skills prowess.

Preoperative Patient Optimization: Outcomes from Elective Total Joint Arthroplasty in a Large US Healthcare System.

INTRODUCTION: Patient factors are known to affect outcomes in arthroplasty surgery. A preoperative program to address modifiable factors may aid in more appropriate patient selection. We sought to assess patient selection and 90-day postoperative outcomes for total joint arthroplasty (TJA) candidates before and after implementation of a preoperative optimization protocol. METHODS: A retrospective time-trend study using data from an integrated healthcare system's total joint replacement registry was done. The study sample consisted of patients aged 18 years or older who underwent primary elective total hip arthroplasty (THA) and total knee arthroplasty (TKA) for osteoarthritis between 2009 and 2019. Patients were divided into two cohorts: procedures performed from 2009 to 2013 ("preoptimization protocol") and those from 2015 to 2019 ("postoptimization protocol"); 2014 was excluded because an evidence-based preoperative patient optimization protocol was progressively implemented throughout the year. Patient characteristics, optimization factors, and 90-day outcomes (including emergency department [ED] visits, readmission, all-cause revision, deep infection, venous thromboembolism, and mortality) were compared between the two cohorts using the Pearson chi-square test or Wilcoxon test. RESULTS: The study sample included 25,925 THA and 60,484 TKA. In the THA cohort, 10,364 (40.0%) and 15,561 (60.0%) were from the preoptimization and postoptimization cohorts, respectively. The postoptimization THA cohort saw lower rates of ED visits (12.1% vs. 9.4%, P < 0.001) and readmissions (5.6% vs. 3.8%, P < 0.001). In the TKA cohort, 24,054 (39.8%) and 36,430 (60.2%) were from the preoptimization and postoptimization cohorts, respectively. The postoptimization TKA cohort saw lower rates of ED visits (13.1% vs. 11.9%, P < 0.001), readmissions (5.6% vs. 3.0%, P < 0.001), all-cause revisions (0.5% vs. 0.3%, P = 0.003), venous thromboembolism (1.3% vs. 0.9%, P < 0.001), and mortality (0.3% vs. 0.2%, P = 0.011). DISCUSSION: An evidence-based protocol to improve patient selection based on modifiable patient factors was adopted by orthopaedic surgeons in the system and utilization of the protocol before TJA was associated with fewer postoperative complications over time. LEVEL OF EVIDENCE: Level IV.

Characteristics Associated With Outstanding General Surgery Residency Graduate Performance, as Rated by Surgical Educators.

Importance: Characteristics of outstanding graduating surgical residents are currently undefined. Identifying these qualities may be important in guiding resident selection and resident education. Objective: To determine characteristics that are most strongly associated with being rated as an outstanding graduating surgical resident. Design, Setting, and Participants: The multi-institutional study had 3 phases. First, an expert panel developed a list of characteristics embodied by top graduating surgical residents. Second, groups of faculty from 14 US general-surgery residency programs ranked 2017 through 2020 graduates into quartiles of overall performance. Third, faculty evaluated their graduates on each characteristic using a 5-point Likert scale. Data were analyzed using Spearman rank-order correlation to identify which individual characteristics were associated with overall graduate performance. A least absolute shrinkage and selection operator (LASSO) ordinal regression was performed to select a parsimonious model to predict the outcome of overall performance rating from individual characteristic scores. Main Outcome and Measures: Surgical educators' rankings of general surgery residency graduates' overall performance. Results: Fifty faculty from 14 US residency programs with a median of 13 (range, 5-30) years of surgical education experience evaluated 297 general surgery residency graduates. Surgical educators identified 21 characteristics that they believed outstanding graduating surgical residents possessed. Two hundred ninety-seven surgical residency graduates were evaluated. Higher scores in every characteristic correlated with better overall performance. Characteristics most strongly associated with higher overall performance scores were surgical judgment (r = 0.728; P < .001), leadership (r = 0.726; P < .001), postoperative clinical skills (r = 0.715; P < .001), and preoperative clinical skills (r = 0.707; P < .001). The remainder of the characteristics were moderately associated with overall performance. The LASSO regression model identified 3 characteristics from which overall resident performance could be accurately predicted without measuring other qualities: surgical judgment (odds ratio [OR] per 1 level of 5-level Likert scale OR, 1.27; 95% CI, 1.03-1.51), leadership (OR, 1.27; 95% CI, 1.06-1.48), and medical knowledge (OR, 1.16; 95% CI, 1.01-1.33). Conclusions and Relevance: All individual characteristics identified by surgical educators as being qualities of outstanding graduating surgical residents were positively associated with overall graduate performance. Surgical judgment and leadership skills had the strongest individual associations. Assessment of only 3 qualities (surgical judgment, leadership, and medical knowledge) were required to predict overall resident performance ratings. These findings highlight the importance of developing specific surgical judgment and leadership skills curricula and assessments during surgical residency.

Defining a Body Mass Index Threshold for Preventing Recurrence in Ventral Hernia Repairs.

INTRODUCTION: Body mass index (BMI) has been established as an independent risk factor for complications after abdominal hernia repairs. While various thresholds have been proposed, there is no consensus for an ideal BMI for elective hernia repair. OBJECTIVE: To identify the BMI threshold at which risk for hernia recurrence is significantly increased in patients undergoing ventral and incisional hernia repair. METHODS: This retrospective review of medical records included patients who underwent ventral or incisional hernia repairs from 2014 to 2020 at a single institution. Patients with hernia defects ≥4 cm were included. The primary outcome measure was hernia recurrence. Classification and Regression Tree (CART) analysis was used to determine the BMI threshold for recurrence. Bivariate and multivariate analyses were used to validate the threshold and to evaluate factors associated with recurrence. RESULTS: Of the 175 patients included, 9.1% had a recurrence. Classification and Regression Tree analysis identified BMI 35.3 kg/m2 as the critical threshold for hernia recurrence. In bivariate analysis, compared to patients who had no recurrence, patients with recurrence were more likely to have cirrhosis (12.5% vs 0%, P = .008), incarcerated hernias (75.0% vs 31.4%, P = .001), urgent surgery (75.0% vs 22.0%, P = <.001), biologic and no mesh use (25.0% vs 6.4% and 12.5% vs 5.7%, P = .012), and BMI >35.3 kg/m2 (75.0% vs 25.8%, P < .001). In multivariate regression, only BMI >35.3 kg/m2 was associated with recurrence [OR: 20.58 (95% CI: 2.17-194.87), P = .008]. CONCLUSION: Body mass index >35.3 kg/m2 was the only independent factor associated with hernia recurrence. This highlights the importance of determining a BMI threshold for patients undergoing ventral or incisional hernia repair.

The impact of the novel coronavirus pandemic on gastrointestinal operative volume in the United States.

BACKGROUND: In March 2020, the Surgeon General recommended limiting elective procedures to prepare for the COVID-19 surge. We hypothesize a consequence of COVID-19 is reduced operative volume across the country. We aim to examine changes in volume of common gastrointestinal operations during COVID-19, including elective, urgent/emergent, and cancer operations. We also evaluate if hospitals with more COVID-19 admissions were most impacted. METHODS: The Vizient database was used to determine monthly operative volume from November 2019 to June 2020 for elective operations (hiatal hernia repairs, bariatric surgery), urgent operations (cholecystectomies, appendectomies, inguinal hernia repairs), and cancer operations (colectomies, gastrectomies, esophagectomies). COVID-19 admissions per hospital were also determined. November 2019-January 2020 was defined as "pre-COVID." The monthly reduction in volume from pre-COVID was calculated for each operation. The top quartile (25%) of hospitals with the most COVID admissions were also evaluated separately from hospitals with fewer COVID cases. Data were analyzed using analysis of variance. RESULTS: Data from 559 hospitals were analyzed. The volumes of all operations evaluated were significantly reduced during the pandemic except gastrectomies and esophagectomies for cancer. The greatest reduction in all operations was in April. In April, the volume of bariatric surgery reduced by 98% (P < 0.001), hiatal hernia repairs by 96% (P < 0.001), urgent cholecystectomies by 42% (P < 0.001), urgent inguinal hernia repairs by 40% (P < 0.001), urgent appendectomies by 24% (P < 0.001), and colectomies for cancer by 39% (P < 0.001). Hospitals with the most COVID-19 admissions had greater reductions in all operations than hospitals with fewer COVID cases. CONCLUSIONS: The coronavirus pandemic led to a significant reduction in volume of all gastrointestinal operations evaluated except gastrectomies and esophagectomies. While elective, non-cancer operations were most affected, urgent and some cancer operations also declined significantly. As COVID-19 continues to surge, Americans may suffer continued limited access to surgical care and a significant operative backlog may be forthcoming.

Outcomes of obese patients hospitalized with COVID-19: the impact of prior bariatric surgery.

BACKGROUND: Obesity and several obesity-related co-morbidities are risk factors for severe COVID-19 disease. Because bariatric surgery successfully treats obesity-related conditions, we hypothesized that prior bariatric surgery may be associated with less severe COVID-19 disease. OBJECTIVES: To examine the association between prior bariatric surgery and outcomes in patients with obesity admitted with COVID-19. SETTING: United States METHODS: The Vizient database was used to obtain demographic and outcomes data for adults with obesity admitted with COVID-19 from May 2020 to January 2021. Patients were divided into 2 groups: those with and those without prior bariatric surgery. The primary outcome was in-hospital mortality. Secondary outcomes were mortality by age, sex, race/ethnicity, and co-morbidity; intubation rate; hemodialysis rate; and length of stay. Because the database only provides aggregate data and not patient-level data, multivariate analysis could not be performed. RESULTS: Among the 124,699 patients with obesity admitted with COVID-19, 2,607 had previous bariatric surgery and 122,092 did not. The proportion of patients ≥65 years of age was higher in the non-bariatric surgery group (36.0% versus 27.6%, P < .0001). Compared with patients without prior bariatric surgery, patients with prior bariatric surgery had lower in-hospital mortality (7.8 versus 11.2%, P < .0001) and intubation rates (18.5% versus 23.6%, P = .0009). Hemodialysis rate (7.2% versus 6.9%, P = .5) and length of stay (8.8 versus 9.6 days, P = .8) were similar between groups. Mortality was significantly lower in the bariatric surgery group for patients 18-64 years of age (5.9% versus 7.4%, P = .01) and ≥65 years of age (12.9% versus 17.9%, P = .0006). CONCLUSIONS: This retrospective cohort study found that inpatients with obesity and COVID-19 who had prior bariatric surgery had improved outcomes compared with a similar cohort without prior bariatric surgery. Further studies should examine mechanisms for the association between bariatric surgery and less severe COVID-19.

Factors Associated With General Surgery Residents' Operative Experience During the COVID-19 Pandemic.

Importance: The suspension of elective operations in March 2020 to prepare for the COVID-19 surge posed significant challenges to resident education. To mitigate the potential negative effects of COVID-19 on surgical education, it is important to quantify how the pandemic influenced resident operative volume. Objective: To examine the association of the pandemic with general surgical residents' operative experience by postgraduate year (PGY) and case type and to evaluate if certain institutional characteristics were associated with a greater decline in surgical volume. Design, Setting, and Participants: This retrospective review included residents' operative logs from 3 consecutive academic years (2017-2018, 2018-2019, and 2019-2020) from 16 general surgery programs. Data collected included total major cases, case type, and PGY. Faculty completed a survey about program demographics and COVID-19 response. Data on race were not collected. Operative volumes from March to June 2020 were compared with the same period during 2018 and 2019. Data were analyzed using Kruskal-Wallis test adjusted for within-program correlations. Main Outcome and Measures: Total major cases performed by each resident during the first 4 months of the pandemic. Results: A total of 1368 case logs were analyzed. There was a 33.5% reduction in total major cases performed in March to June 2020 compared with 2018 and 2019 (45.0 [95% CI, 36.1-53.9] vs 67.7 [95% CI, 62.0-72.2]; P < .001), which significantly affected every PGY. All case types were significantly reduced in 2020 except liver, pancreas, small intestine, and trauma cases. There was a 10.2% reduction in operative volume during the 2019-2020 academic year compared with the 2 previous years (192.3 [95% CI, 178.5-206.1] vs 213.8 [95% CI, 203.6-223.9]; P < .001). Level 1 trauma centers (49.5 vs 68.5; 27.7%) had a significantly lower reduction in case volume than non-level 1 trauma centers (33.9 vs 63.0; 46%) (P = .03). Conclusions and Relevance: In this study of operative logs of general surgery residents in 16 US programs from 2017 to 2020, the first 4 months of the COVID-19 pandemic was associated with a significant reduction in operative experience, which affected every PGY and most case types. Level 1 trauma centers were less affected than non-level 1 centers. If this trend continues, the effect on surgical training may be even more detrimental.

Intracystic Hemorrhage Complicating Polycystic Liver Disease in a 90-Year-Old Woman.

Nonparasitic hepatic cysts are common benign tumors that are often asymptomatic and incidentally discovered on imaging. Intracystic hemorrhage is a rare complication of hepatic cysts. We review the literature and discuss a case of intracystic hemorrhage in a 90-year-old woman with polycystic liver disease. The patient underwent cyst aspiration and percutaneous drain placement with subsequent resolution of symptoms. To our knowledge, we report the oldest patient to present with hemorrhage into a hepatic cyst. This case presents unique challenges in management, both because of the patient's age and because of her polycystic liver disease.

Procedural simulation: medical student preference and value of three task trainers for ultrasound guided regional anesthesia.

BACKGROUND: Ultrasound guided regional anesthesia is widely taught using task trainer models. Commercially available models are often used; however, they can be cost prohibitive. Therefore, alternative "homemade" models with similar fidelity are often used. We hypothesize that professional task trainers will be preferred over homemade models. The purpose of this study is to determine realism, durability and cleanliness of three different task trainers for ultrasound guided nerve blocks. METHODS: This was a prospective observational study using a convenience sample of medical student participants in an ultrasound guided nerve block training session on January 24th, 2015. Participants were asked to perform simulated nerve blocks on three different task trainers including, 1 commercial and 2 homemade. A questionnaire was then given to all participants to rate their experiences both with and without the knowledge on the cost of the simulator device. RESULTS: Data was collected from 25 participants. The Blue Phantom model was found to have the highest fidelity. Initially, 10 (40%) of the participants preferred the Blue Phantom model, while 10 (40%) preferred the homemade gelatin model and 5 (20%) preferred the homemade tofu model. After cost awareness, the majority, 18 (72%) preferred the gelatin model. CONCLUSION: The Blue Phantom model was thought to have the highest fidelity, but after cost consideration the homemade gelatin model was preferred.

Dr Google: The readability and accuracy of patient education websites for Graves' disease treatment.

BACKGROUND: National guidelines emphasize the importance of incorporating patient preferences into the recommendations for the treatment of Graves' disease. Many patients use the Internet to obtain health information, and search results can affect their treatment decisions. This study compares the readability and accuracy of patient-oriented online resources for the treatment of Graves' disease by website affiliation and treatment modality. METHODS: A systematic Internet search was used to identify the top websites discussing the treatment of Graves' disease. Readability was measured using 5 standardized tests. Accuracy was assessed by a blinded, expert panel, which scored the accuracy of sites on a scale of 1 to 5. Mean readability and accuracy scores were compared among website affiliations and treatment modalities. RESULTS: We identified 13 unique websites, including 2 academic, 2 government, 5 nonprofit, and 4 private sites. There was a difference in both readability (mean 13.2, range 9.1-15.7, P = .003) and accuracy (mean 4.04, range 2.75-4.50, P = .019) based on website affiliation. Government sites (mean readability 11.1) were easier to read than academic (14.3, P < .01), nonprofit (13.9, P < .01), and private sites (13.5, P < .05). Academic sites (mean accuracy 4.50) were more accurate than private sites (3.56, P < .05). CONCLUSION: Online patient resources for the treatment of Graves' disease are written at an inappropriately high reading level. Academic sites contain both the most accurate and the most difficult to read information. Private sites represented the majority of our top results but contained the least accurate information.

Procedural simulation: medical student preference and value of three task trainers for ultrasound guided regional anesthesia / 世界急诊医学杂志（英文）

@#BACKGROUND: Ultrasound guided regional anesthesia is widely taught using task trainer models. Commercially available models are often used; however, they can be cost prohibitive. Therefore, alternative "homemade" models with similar fidelity are often used. We hypothesize that professional task trainers will be preferred over homemade models. The purpose of this study is to determine realism, durability and cleanliness of three different task trainers for ultrasound guided nerve blocks. METHODS: This was a prospective observational study using a convenience sample of medical student participants in an ultrasound guided nerve block training session on January 24th, 2015. Participants were asked to perform simulated nerve blocks on three different task trainers including, 1 commercial and 2 homemade. A questionnaire was then given to all participants to rate their experiences both with and without the knowledge on the cost of the simulator device. RESULTS: Data was collected from 25 participants. The Blue Phantom model was found to have the highest fidelity. Initially, 10 (40%) of the participants preferred the Blue Phantom model, while 10 (40%) preferred the homemade gelatin model and 5 (20%) preferred the homemade tofu model. After cost awareness, the majority, 18 (72%) preferred the gelatin model. CONCLUSION: The Blue Phantom model was thought to have the highest fidelity, but after cost consideration the homemade gelatin model was preferred.

Feasibility study of first-year medical students identifying cardiac anatomy using ultrasound in rural Panama.

BACKGROUND: There are over 15 million children who have cardiac anomalies around the world, resulting in a significant morbidity and mortality. Early recognition and treatment can improve the outcomes and lengthen life-expectancy of these patients. The NIH and WHO have promoted guidelines for screening for congenital cardiac anomalies using ultrasound in rural environments. METHODS: Our study took place in Bocas Del Toro, Panama where a mobile clinic was established for community healthcare screening and ultrasonographic evaluation by medical student volunteers and volunteer clinical faculty. This was a non-blinded, investigational study utilizing a convenience sample of pediatric patients presenting for voluntary evaluation. Seven first-year medical students were recruited for the study. These students underwent a training program for advanced cardiac ultrasound instruction, termed "Pediatric Echocardiography Cardiac Screening (PECS)". RESULTS: Ten patients were enrolled in the study. Nine patients had adequate images as defined by the PECS criteria and were all classified as normal cardiac pathology by the medical students, resulting in a sensitivity and specificity of 100%. A single patient was identified by medical students as having a pathologic pulmonic stenosis. This was confirmed as correct by a blinded ultrasonographer. CONCLUSIONS: In this pilot study, the first-year medical students were able to correctly identify pediatric cardiac anatomy and pathology in rural Panama after undergoing a 12-hour ultrasound PECS training session. We believe that with this knowledge, minimally trained practitioners can be used to screen for cardiac anomalies in rural Panama using ultrasound.

Feasibility study of first-year medical students identifying cardiac anatomy using ultrasound in rural Panama / 世界急诊医学杂志（英文）

BACKGROUND: There are over 15 million children who have cardiac anomalies around the world, resulting in a significant morbidity and mortality. Early recognition and treatment can improve the outcomes and lengthen life-expectancy of these patients. The NIH and WHO have promoted guidelines for screening for congenital cardiac anomalies using ultrasound in rural environments. METHODS: Our study took place in Bocas Del Toro, Panama where a mobile clinic was established for community healthcare screening and ultrasonographic evaluation by medical student volunteers and volunteer clinical faculty. This was a non-blinded, investigational study utilizing a convenience sample of pediatric patients presenting for voluntary evaluation. Seven first-year medical students were recruited for the study. These students underwent a training program for advanced cardiac ultrasound instruction, termed "Pediatric Echocardiography Cardiac Screening (PECS)". RESULTS: Ten patients were enrolled in the study. Nine patients had adequate images as defined by the PECS criteria and were all classified as normal cardiac pathology by the medical students, resulting in a sensitivity and specificity of 100％. A single patient was identified by medical students as having a pathologic pulmonic stenosis. This was confirmed as correct by a blinded ultrasonographer. CONCLUSIONS: In this pilot study, the first-year medical students were able to correctly identify pediatric cardiac anatomy and pathology in rural Panama after undergoing a 12-hour ultrasound PECS training session. We believe that with this knowledge, minimally trained practitioners can be used to screen for cardiac anomalies in rural Panama using ultrasound.

The ap-2 clathrin adaptor mediates endocytosis of an inhibitory killer cell Ig-like receptor in human NK cells.

Stable surface expression of human inhibitory killer cell Ig-like receptors (KIRs) is critical for controlling NK cell function and maintaining NK cell tolerance toward normal MHC class I(+) cells. Our recent experiments, however, have found that Ab-bound KIR3DL1 (3DL1) readily leaves the cell surface and undergoes endocytosis to early/recycling endosomes and subsequently to late endosomes. We found that 3DL1 internalization is at least partially mediated by an interaction between the µ2 subunit of the AP-2 clathrin adaptor complex and ITIM tyrosine residues in the cytoplasmic domain of 3DL1. Disruption of the 3DL1/µ2 interaction, either by mutation of the ITIM tyrosines in 3DL1 or mutation of µ2, significantly diminished endocytosis and increased surface expression of 3DL1 in human primary NK cells and cell lines. Furthermore, we found that the 3DL1/AP-2 interaction is diminished upon Ab engagement with the receptor, as compared with untreated cells. Thus, we have identified AP-2-mediated endocytosis as a mechanism regulating the surface levels of inhibitory KIRs through their ITIM domains. Based on our results, we propose a model in which nonengaged KIRs are internalized by this mechanism, whereas engagement with MHC class I ligand would diminish AP-2 binding, thereby prolonging stable receptor surface expression and promoting inhibitory function. Furthermore, this ITIM-mediated mechanism may similarly regulate the surface expression of other inhibitory immune receptors.

Ubiquitylation of an internalized killer cell Ig-like receptor by Triad3A disrupts sustained NF-κB signaling.

Killer cell Ig-like receptor (KIR) with two Ig-like domains and a long cytoplasmic domain 4 (2DL4; CD158d) is a unique KIR expressed on human NK cells, which stimulates cytokine production, but mechanisms regulating its expression and function are poorly understood. By yeast two-hybrid screening, we identified the E3 ubiquitin ligase, Triad3A, as an interaction partner for the 2DL4 cytoplasmic domain. The protein interaction was confirmed in vivo, and Triad3A expression induced polyubiquitylation and degradation of 2DL4. Overexpression of Triad3A selectively abrogated the cytokine-producing function of 2DL4, whereas Triad3A short hairpin RNA reversed ubiquitylation and restored cytokine production. Expression of Triad3A in an NK cell line did not affect receptor surface expression, internalization, or early signaling, but significantly reduced receptor turnover and suppressed sustained NF-κB activation. 2DL4 endocytosis was found to be vital to stimulate cytokine production, and Triad3A expression diminished localization of internalized receptor in early endosomes. Our results reveal a critical role for endocytosed 2DL4 receptor to generate sustained NF-κB signaling and drive cytokine production. We conclude that Triad3A is a key negative regulator of sustained 2DL4-mediated NF-κB signaling from internalized 2DL4, which functions by promoting ubiquitylation and degradation of endocytosed receptor from early endosomes.

Structure/function of human killer cell immunoglobulin-like receptors: lessons from polymorphisms, evolution, crystal structures and mutations.

Stimulation or tolerance of natural killer (NK) cells is achieved through a cross-talk of signals derived from cell surface activating and inhibitory receptors. Killer cell immunoglobulin-like receptors (KIR) are a family of highly polymorphic activating and inhibitory receptors that serve as key regulators of human NK cell function. Distinct structural domains in different KIR family members determine function by providing docking sites for ligands or signalling proteins. Here, we review a growing body of literature that has identified important structural elements on KIR that contribute to function through studies of engineered mutants, natural polymorphic sequence variants, crystal structure data and the conservation of protein sequences throughout primate evolution. Extensive natural polymorphism is associated with both human KIR and their ligands, MHC class I (HLA-A, -B and -C) molecules, and numerous studies have demonstrated associations between inheritance of certain combinations of KIR and HLA genes and susceptibility to several diseases, including viral infections, autoimmune disorders and cancers. In addition, certain KIR/HLA combinations can influence pregnancy and the outcome of haematopoietic stem cell transplantation. In view of the significant regulatory influences of KIR on immune function and human health, it is essential to fully understand the impacts of these polymorphic sequence variations on ligand recognition, expression and function of the receptor.

Introduction of shRNAs into human NK-like cell lines with retrovirus.

Natural killer (NK) cell lines are difficult to transfect using standard techniques, which limits the ability to establish long-term knockdown of proteins with short-hairpin (sh)RNAs. We have developed a method to stably knockdown protein expression in human NK-like lines by introducing shRNAs in retroviral vectors. After a single transduction with retrovirus, shRNA-containing cells can be selected with drug treatment or sorted for enhanced green fluorescent protein (EGFP) expression. With this method, protein expression can be stably decreased to less than 10% of wild-type levels.