The chaperone PrsA2 regulates the secretion, stability, and folding of listeriolysin O during Listeria monocytogenes infection.

Pathogenic bacteria rely on secreted virulence factors to cause disease in susceptible hosts. However, in Gram-positive bacteria, the mechanisms underlying secreted protein activation and regulation post-membrane translocation remain largely unknown. Using proteomics, we identified several proteins that are dependent on the secreted chaperone PrsA2. We followed with phenotypic, biochemical, and biophysical assays and computational analyses to examine the regulation of a detected key secreted virulence factor, listeriolysin O (LLO), and its interaction with PrsA2 from the bacterial pathogen Listeria monocytogenes (Lm). Critical to Lm virulence is internalization by host cells and the subsequent action of the cholesterol-dependent pore-forming toxin, LLO, which enables bacterial escape from the host cell phagosome. Since Lm is a Gram-positive organism, the space between the cell membrane and wall is solvent exposed. Therefore, we hypothesized that the drop from neutral to acidic pH as the pathogen is internalized into a phagosome is critical to regulating the interaction of PrsA2 with LLO. Here, we demonstrate that PrsA2 directly interacts with LLO in a pH-dependent manner. We show that PrsA2 protects and sequesters LLO under neutral pH conditions where LLO can be observed to aggregate. In addition, we identify molecular features of PrsA2 that are required for interaction and ultimately the folding and activity of LLO. Moreover, protein-complex modeling suggests that PrsA2 interacts with LLO via its cholesterol-binding domain. These findings highlight a mechanism by which a Gram-positive secretion chaperone regulates the secretion, stability, and folding of a pore-forming toxin under conditions relevant to host cell infection. IMPORTANCE: Lm is a ubiquitous food-borne pathogen that can cause severe disease to vulnerable populations. During infection, Lm relies on a wide repertoire of secreted virulence factors including the LLO that enables the bacterium to invade the host and spread from cell to cell. After membrane translocation, secreted factors must become active in the challenging bacterial cell membrane-wall interface. However, the mechanisms required for secreted protein folding and function are largely unknown. Lm encodes a chaperone, PrsA2, that is critical for the activity of secreted factors. Here, we show that PrsA2 directly associates and protects the major Lm virulence factor, LLO, under conditions corresponding to the host cytosol, where LLO undergoes irreversible denaturation. Additionally, we identify molecular features of PrsA2 that enable its interaction with LLO. Together, our results suggest that Lm and perhaps other Gram-positive bacteria utilize secreted chaperones to regulate the activity of pore-forming toxins during infection.

Association of Women Surgeons' Comprehensive Initiative for Healthy Surgical Families During Residency and Fellowship Training.

Importance: The lack of family-friendly policies continues to contribute to the underrepresentation and attrition of surgical trainees. Women in surgery face unique challenges in balancing surgical education with personal and family needs. Observations: The Association of Women Surgeons is committed to supporting surgical families and developing equitable family-friendly guidelines. Herein we detail recommendations for adequate paid parental leave, access to childcare, breastfeeding support, and insurance coverage of fertility preservation and assisted reproductive technology. Conclusions and Relevance: The specific recommendations outlined in this document form the basis of a comprehensive initiative for supporting surgical families.

Does the American College of Surgeons National Surgical Quality Improvement Program (ACS NSQIP) accurately predict mortality for patients with elevated MELD scores?

BACKGROUND: The Model for End Stage Liver Disease (MELD) predicts mortality for liver disease patients. The American College of Surgeons National Surgical Quality Improvement Program (NSQIP) estimates mortality risk for surgical patients; however, NSQIP does not collect data regarding liver disease. This study's aim was to examine the accuracy of NSQIP mortality estimates for patients with elevated MELD scores. METHODS: NSQIP participant user files from 2005 to 2016 were queried. MELD scores were calculated and patients with scores ≥10 included. NSQIP-predicted mortality was compared to actual mortality. RESULTS: 268,873 patients met inclusion criteria. Predicted and observed number of 30-day postoperative deaths were 20,644 (7.7%) and 21,764 (8.1%). For patients with MELD ≥24, NSQIP-predicted 30-day mortality underestimated actual mortality. For patients with MELD ≤22, predicted and actual risks were similar. CONCLUSION: NSQIP predicts 30-day mortality risk well for patients with MELD scores from 10 to 22, but underestimates risk for patients with higher MELD scores.

Undertreatment of Pancreatic Cancer: The Intersection of Bias, Biology, and Geography.

Pancreatic cancer is the third leading cause of cancer deaths in the United States. Black patients with pancreatic cancer experience higher incidence and increased mortality. Although racial biologic differences exist, socioeconomic status, insurance type, physician bias, and patient beliefs contribute to the disparities in outcomes observed among patients who are Black, indigenous, and people of color.

CAR T cells targeting tumor-associated exons of glypican 2 regress neuroblastoma in mice.

Targeting solid tumors must overcome several major obstacles, in particular, the identification of elusive tumor-specific antigens. Here, we devise a strategy to help identify tumor-specific epitopes. Glypican 2 (GPC2) is overexpressed in neuroblastoma. Using RNA sequencing (RNA-seq) analysis, we show that exon 3 and exons 7-10 of GPC2 are expressed in cancer but are minimally expressed in normal tissues. Accordingly, we discover a monoclonal antibody (CT3) that binds exons 3 and 10 and visualize the complex structure of CT3 and GPC2 by electron microscopy. The potential of this approach is exemplified by designing CT3-derived chimeric antigen receptor (CAR) T cells that regress neuroblastoma in mice. Genomic sequencing of T cells recovered from mice reveals the CAR integration sites that may contribute to CAR T cell proliferation and persistence. These studies demonstrate how RNA-seq data can be exploited to help identify tumor-associated exons that can be targeted by CAR T cell therapies.

Ataxia telangiectasia mutated germline pathogenic variant in adrenocortical carcinoma.

BACKGROUND: Adrenocortical carcinoma (ACC) is a rare malignancy arising from the adrenal cortex. ACC carries a dismal prognosis and surgery offers the only chance for a cure. Germline pathogenic variants among certain oncogenes have been implicated in ACC. Here, we report the first case of ACC in a patient with a pathogenic variant in the Ataxia Telangiectasia Mutated (ATM) gene. PATIENTS AND METHODS: A 56-year-old Caucasian woman with biopsy proven ACC deemed unresectable and treated with etoposide, doxorubicin and cisplatin (EDP), and mitotane presented to our institution for evaluation. The tumor specimen was examined pathologically, and genetic analyses were performed on the tumor and germline using next-generation sequencing. RESULTS: Pathologic evaluation revealed an 18.0 × 14.0 × 9.0 cm low-grade ACC with tumor free resection margins. Immunohistochemistry stained for inhibin, melan-A, and chromogranin. ClinOmics analysis revealed a germline pathogenic deletion mutation of one nucleotide in ATM is denoted as c.1215delT at the cDNA level and p.Asn405LysfsX15 (N405KfsX15) at the protein level. Genomic analysis of the tumor showed loss of heterozygosity (LOH) of chromosome 11 on which the ATM resides. CONCLUSION: ACC is an aggressive malignancy for which surgical resection currently offers the only curative option. Here we report a heterozygous loss-of-function mutation in germline DNA and LOH of ATM in tumor in an ACC patient, a classic two-hit scenario in a well-known cancer suppresser gene, suggesting a pathogenic role of the ATM gene in certain ACC cases.

Pathologic upstaging in resected pancreatic adenocarcinoma: Risk factors and impact on survival.

BACKGROUND: Clinical and pathologic staging determine treatment of pancreatic cancer. Clinical stage has been shown to underestimate final pathologic stage in pancreatic cancer, resulting in upstaging. METHODS: National Cancer Database was used to identify clinical stage I pancreatic adenocarcinoma. Univariate, multivariable logistic regression, and Cox proportional hazard ratio were used to determine differences between upstaged and stage concordant patients. RESULTS: Upstaging was seen in 80.2% of patients. Factors found to be significantly associated with upstaging included pancreatic head tumors (OR 2.56), high-grade histology (OR 1.74), elevated Ca 19-9 (OR 2.09), and clinical stage T2 (OR 1.99). Upstaging was associated with a 45% increased risk of mortality compared to stage concordant disease (HR 1.44, p < .001). CONCLUSION: A majority of clinical stage I pancreatic cancer is upstaged after resection. Factors including tumor location, grade, Ca 19-9, and tumor size can help identify those at high risk for upstaging.

Adjuvant Chemotherapy After Neoadjuvant Chemotherapy for Pancreatic Cancer is Associated with Improved Survival for Patients with Low-Risk Pathology.

BACKGROUND: With limited evidence, the benefit of adjuvant chemotherapy (AT) after completion of neoadjuvant chemotherapy (NT) and surgical resection for patients with pancreatic adenocarcinoma is debated. Guidelines recommend 6 months of AT for patients receiving NT. However, the patient-derived benefit from additional AT remains unknown. METHODS: The National Cancer Database from 2006 to 2015 was used to identify patients undergoing NT. The chi-square test and multivariable logistic regression were used to identify differences between those receiving only NT and those receiving NT and AT. Survival analysis using the Kaplan-Meier method and the Cox proportional hazard ratio model was applied to the entire cohort and to subgroups with differing lymph node ratios (LNRs), tumor sizes, grades, and surgical margin statuses. RESULTS: Of the 3897 patients who received NT, 36.7 % received additional AT. Analysis of the entire cohort showed that associated survival was significantly improved with NT and AT compared with NT alone (hazard ratio [HR], 0.83; p < 0.001). In the subgroup analysis, the survival benefit of additional AT remained significant for those with negative nodal disease, an LNR lower than 0.15, low-grade histology, and negative margin status. Overall survival did not differ between those receiving NT only and those receiving NT and AT in the group with an LNR of 0.15 or higher, high-grade histology, and positive margins. CONCLUSION: This study identified an increasing trend in the use of AT after NT and showed an associated survival benefit for subgroups with low-risk pathologic features. These results suggest that the addition of AT after NT likely beneficial for these subgroups.

Venous thromboembolism prevention compliance: A multidisciplinary educational approach utilizing NSQIP best practice guidelines.

BACKGROUND: Review of our institutional National Surgical Quality Improvement Project (NSQIP) data found higher rate of Venous Thromboembolic Events (VTE) (2.5% vs. 1.1%). Compared to the national benchmark. Our goal was to identify opportunities for quality improvement. METHODS: We compared NSQIP general surgery data from January 2015-December 2016 (period 1) to January 2017-December 2018 (period 2). A multidisciplinary committee was developed and patient centered education implemented to enhance VTE compliance. RESULTS: Over 50% of all the patients who developed VTE were non-compliant with chemical prophylaxis. The majority of non-compliance was due to pain. During period 1 there were 12 VTEs in 482 cases, while in period two, 18 VTEs in 2347 cases (2.5% vs. 0.8%; RR 2.3, 95% CI 1.5-3.7, p < 0.001). Missed chemical prophylaxis decreased from 50 to 17 per week after the intervention. CONCLUSION: A multidisciplinary, patient centered approach to increase VTE prevention decreases VTE rates to below a comparable benchmark.

Persistent Polyfunctional Chimeric Antigen Receptor T Cells That Target Glypican 3 Eliminate Orthotopic Hepatocellular Carcinomas in Mice.

BACKGROUND AND AIMS: Glypican 3 (GPC3) is an oncofetal antigen involved in Wnt-dependent cell proliferation that is highly expressed in hepatocellular carcinoma (HCC). We investigated whether the functions of chimeric antigen receptors (CARs) that target GPC3 are affected by their antibody-binding properties. METHODS: We collected peripheral blood mononuclear cells from healthy donors and patients with HCC and used them to create CAR T cells, based on the humanized YP7 (hYP7) and HN3 antibodies, which have high affinities for the C-lobe and N-lobe of GPC3, respectively. NOD/SCID/IL-2Rgcnull (NSG) mice were given intraperitoneal injections of luciferase-expressing (Luc) Hep3B or HepG2 cells and after xenograft tumors formed, mice were given injections of saline or untransduced T cells (mock control), or CAR (HN3) T cells or CAR (hYP7) T cells. In other NOD/SCID/IL-2Rgcnull (NSG) mice, HepG2-Luc or Hep3B-Luc cells were injected into liver, and after orthotopic tumors formed, mice were given 1 injection of CAR (hYP7) T cells or CD19 CAR T cells (control). We developed droplet digital polymerase chain reaction and genome sequencing methods to analyze persistent CAR T cells in mice. RESULTS: Injections of CAR (hYP7) T cells eliminated tumors in 66% of mice by week 3, whereas CAR (HN3) T cells did not reduce tumor burden. Mice given CAR (hYP7) T cells remained tumor free after re-challenge with additional Hep3B cells. The CAR T cells induced perforin- and granzyme-mediated apoptosis and reduced levels of active ß-catenin in HCC cells. Mice injected with CAR (hYP7) T cells had persistent expansion of T cells and subsets of polyfunctional CAR T cells via antigen-induced selection. These T cells were observed in the tumor microenvironment and spleen for up to 7 weeks after CAR T-cell administration. Integration sites in pre-infusion CAR (HN3) and CAR (hYP7) T cells were randomly distributed, whereas integration into NUPL1 was detected in 3.9% of CAR (hYP7) T cells 5 weeks after injection into tumor-bearing mice and 18.1% of CAR (hYP7) T cells at week 7. There was no common site of integration in CAR (HN3) or CD19 CAR T cells from tumor-bearing mice. CONCLUSIONS: In mice with xenograft or orthoptic liver tumors, CAR (hYP7) T cells eliminate GPC3-positive HCC cells, possibly by inducing perforin- and granzyme-mediated apoptosis or reducing Wnt signaling in tumor cells. GPC3-targeted CAR T cells might be developed for treatment of patients with HCC.

Gastric Cancer Treatments and Survival Trends in the United States.

Gastric cancer is the third most common cause of cancer deaths worldwide. Despite evidence-based recommendation for treatment, the current treatment patterns for all stages of gastric cancer remain largely unexplored. This study investigates trends in the treatments and survival of gastric cancer. The National Cancer Database was used to identify gastric adenocarcinoma patients from 2004-2016. Chi-square tests were used to examine subgroup differences between disease stages: Stage I, II/III and IV. Multivariate analyses identified factors associated with the receipt of guideline concordant care. The Kaplan-Meier method was used to assess three-year overall survival. The final cohort included 108,150 patients: 23,584 Stage I, 40,216 Stage II/III, and 44,350 Stage IV. Stage specific guideline concordant care was received in only 73% of patients with Stage I disease and 51% of patients with Stage II/III disease. Patients who received guideline consistent care had significantly improved survival compared to those who did not. Overall, we found only moderate improvement in guideline adherence and three-year overall survival during the 13-year study time period. This study showed underutilization of stage specific guideline concordant care for stage I and II/III disease.

Recognizing and Reacting to Microaggressions in Medicine and Surgery.

Diversity and inclusion in medicine, and in surgery in particular, still merit substantial attention in 2019. With each increase in academic rank there are fewer women, with only 24% of full professors in medicine being women. Underrepresented minorities face similar challenges, with only 3% of medical faculty being black and 4% of medical faculty being Hispanic or Latino; only 2% of full professors are Hispanic or Latino and only another 2% are black. Explicit discrimination unfortunately still does exist, but in many environments, more subtle forms of bias are more prevalent. Microaggressions, which are categorized as microassaults, microinsults, microinvalidations, and environmental microaggressions, are indirect expressions of prejudice that contribute to the maintenance of existing power structures and may limit the hiring, promotion, and retention of women and underrepresented minorities. The primary goal of this communication is to help readers understand microaggressions and their effect. We also provide suggestions for how recipients or bystanders may respond to microaggressions.

Postbiliary drainage rates of cholangitis are impacted by procedural technique for patients with supra-ampullary cholangiocarcinoma: A SEER-Medicare analysis.

BACKGROUND: The optimal approach to biliary drainage for patients with supra-ampullary cholangiocarcinoma remains undetermined. Violation of sphincter of Oddi results in bacterial colonization of bile ducts and may increase postdrainage infectious complications. We sought to determine if rates of cholangitis are affected by the type of drainage procedure. METHODS: We examined the Surveillance, Epidemiology, and End Results-Medicare linked database from 1991 to 2013 for cholangiocarcinoma. Biliary drainage procedures were categorized as sphincter of Oddi violating (SOV) or sphincter of Oddi preserving (SOP). Patients were stratified by resection. RESULTS: A total of 1914 patients were included in the final analysis. A total of 1264 patients did not undergo a postdrainage resection (SOP 83, SOV 1181) while 650 did undergo a postdrainage resection (SOP 26, SOV 624). For those patients not undergoing a postdrainage resection, the rate of cholangitis 90 days after an SOP procedure was 19% compared with 34% in the SOV cohort (P = 0.007). For those patients undergoing a postdrainage resection, the rate of cholangitis 90 days after an SOP procedure was less than 42.3% compared with 30% in the SOV cohort (P = 0.66). CONCLUSION: For patients with supra-ampullary cholangiocarcinoma that did not undergo resection, biliary drainage procedures that violated the sphincter of Oddi were associated with increased rates of cholangitis.

Prolonged Antibiotics for Drains After Spine Injury Instrumentation for Trauma: Not Prophylactic or Necessary.

BACKGROUND: Antibiotics after spine instrumentation are often extended while the surgical drain is in place, particularly for traumatic injuries. We sought to study if continuing antibiotics past 24 hours affected outcomes. METHODS: We performed a retrospective observational study of all patients who underwent spine fixation with hardware and surgical drains for trauma at our institution. We compared the effect of perioperative (≤24 hours of antibiotics) versus prolonged (>24 hours) antibiotics on surgical outcomes. Bivariate and multivariable logistic and linear regression statistics were performed. RESULTS: Three hundred and forty-six patients were included in the analysis. On multivariate analysis, antibiotic duration >24 hours did not predict surgical site infection (odds ratio, 2.68; 95% confidence interval, 0.88-8.10, P = 0.08) or mortality (odds ratio, 0.59; 95% confidence interval, 0.10-3.44; P = 0.56). CONCLUSIONS: Continuing antibiotics past 24 hours after traumatic spine instrumentation was not associated with improved outcomes. A prospective study to verify these findings may be warranted.