Heparin activation of protein Z-dependent protease inhibitor (ZPI) allosterically blocks protein Z activation through an extended heparin-binding site.

Protein Z (PZ)-dependent protease inhibitor (ZPI) is a plasma anticoagulant protein of the serpin superfamily, which is activated by its cofactor, PZ, to rapidly inhibit activated factor X (FXa) on a procoagulant membrane surface. ZPI is also activated by heparin to inhibit free FXa at a physiologically significant rate. Here, we show that heparin binding to ZPI antagonizes PZ binding to and activation of ZPI. Virtual docking of heparin to ZPI showed that a heparin-binding site near helix H close to the PZ-binding site as well as a previously mapped site in helix C was both favored. Alanine scanning mutagenesis of the helix H and helix C sites demonstrated that both sites were critical for heparin activation. The binding of heparin chains 72 to 5-saccharides in length to ZPI was similarly effective in antagonizing PZ binding and in inducing tryptophan fluorescence changes in ZPI. Heparin binding to variant ZPIs with either the helix C sites or the helix H sites mutated showed that heparin interaction with the higher affinity helix C site most distant from the PZ-binding site was sufficient to induce these tryptophan fluorescence changes. Together, these findings suggest that heparin binding to a site on ZPI extending from helix C to helix H promotes ZPI inhibition of FXa and allosterically antagonizes PZ binding to ZPI through long-range conformational changes. Heparin antagonism of PZ binding to ZPI may serve to spare limiting PZ and allow PZ and heparin cofactors to target FXa at different sites of action.

Paramount Importance of Core Conformational Changes for Heparin Allosteric Activation of Antithrombin.

Antithrombin is unique among serpin family protein protease inhibitors with respect to the major reactive center loop (RCL) and core conformational changes that mediate allosteric activation of its anticoagulant function by heparin. A critical role for expulsion of the RCL hinge from a native stabilizing interaction with the hydrophobic core in the activation mechanism has been proposed from reports that antithrombin variants that block this change through engineered disulfide bonds block activation. However, the sufficiency of core conformational changes for activation without expulsion of the RCL from the core is suggested by variants that are activated without the need for heparin and retain the native RCL-core interaction. To resolve these apparently conflicting findings, we engineered variants in which disulfides designed to block the RCL conformational change were combined with constitutively activating mutations. Our findings demonstrate that while a reversible constitutive activation can be engineered in variants that retain the native RCL-core interaction, engineered disulfides that lock the RCL native conformation can also block heparin allosteric activation. Such findings support a three-state allosteric activation model in which constitutive activating mutations stabilize an intermediate-activated state wherein core conformational changes and a major activation have occurred without the release of the RCL from the core but with a necessary repositioning of the RCL to allow productive engagement with an exosite. Rigid disulfide bonds that lock the RCL native conformation block heparin activation by preventing both RCL repositioning in the intermediate-activated state and the release of the RCL from the core in the fully activated state.

Protein Z-dependent protease inhibitor (ZPI) is a physiologically significant inhibitor of prothrombinase function.

Current thought holds that factor Xa (FXa) bound in the prothrombinase complex is resistant to regulation by protein protease inhibitors during prothrombin activation. Here we provide evidence that, contrary to this view, the FXa-specific serpin inhibitor, protein Z-dependent protease inhibitor (ZPI), complexed with its cofactor, protein Z (PZ), functions as a physiologically significant inhibitor of prothrombinase-bound FXa during prothrombin activation. Kinetics studies showed that the rapid rate of inhibition of FXa by the ZPI-PZ complex on procoagulant membrane vesicles (ka(app) ∼107 m-1 s-1) was decreased ∼10-fold when FXa was bound to FVa in prothrombinase and a further ∼3-4-fold when plasma levels of S195A prothrombin were present (ka(app) 2 × 105 m-1 s-1). Nevertheless, the ZPI-PZ complex produced a major inhibition of thrombin generation during prothrombinase-catalyzed activation of prothrombin under physiologically relevant conditions. The importance of ZPI-PZ complex anticoagulant regulation of FXa both before and after incorporation into prothrombinase was supported by thrombin generation assays in plasma. These showed enhanced thrombin generation when the inhibitor was neutralized with a PZ-specific antibody and decreased thrombin generation when exogenous ZPI-PZ complex was added whether prothrombin was activated directly by FXa or through extrinsic or intrinsic pathway activators. Moreover, the PZ antibody enhanced thrombin generation both in the absence and presence of activated protein C (APC) anticoagulant activity. Taken together, these results suggest an important anticoagulant role for the ZPI-PZ complex in regulating both free FXa generated in the initiation phase of coagulation as well as prothrombinase-bound FXa in the propagation phase that complement prothrombinase regulation by APC.

Cooperative Interactions of Three Hotspot Heparin Binding Residues Are Critical for Allosteric Activation of Antithrombin by Heparin.

Heparin allosterically activates the anticoagulant serpin, antithrombin, by binding through a sequence-specific pentasaccharide and inducing activating conformational changes in the protein. Three basic residues of antithrombin, Lys114, Lys125, and Arg129, have been shown to be hotspots for binding the pentasaccharide, but the molecular basis for such hotspot binding has been unclear. To determine whether this results from cooperative interactions, we analyzed the effects of single, double, and triple mutations of the hotspot residues on pentasaccharide binding and activation of antithrombin. Double-mutant cycles revealed that the contribution of each residue to pentasaccharide binding energy was progressively reduced when one or both of the other residues were mutated, indicating strong coupling between each pair of residues that was dependent on the third residue and reflective of the three residues acting as a cooperative unit. Rapid kinetic studies showed that the hotspot residue mutations progressively abrogated the ability of the pentasaccharide to bind productively to native antithrombin and to conformationally activate the serpin by engaging the hotspot residues in an induced-fit interaction. Examination of the antithrombin-pentasaccharide complex structure revealed that the hotspot residues form two adjoining binding pockets for critical sulfates of the pentasaccharide that structurally link these residues. Together, these findings demonstrate that cooperative interactions of Lys114, Lys125, and Arg129 are critical for the productive induced-fit binding of the heparin pentasaccharide to antithrombin that allosterically activates the anticoagulant function of the serpin.

Pancreaticoduodenectomy and metastasectomy for metastatic pancreatic neuroendocrine tumors.

BACKGROUND AND OBJECTIVES: Various treatment options exist for patients with metastatic pancreatic neuroendocrine tumors (PNETs). Surgical resection with pancreaticoduodenectomy (PD) typically reserved for patients with limited disease. Definitive data are lacking to support either the resection of primary PNET in the metastatic setting or for surgical debulking of metastatic lesions. METHODS: We conducted an analysis of the National Cancer Database (NCDB) using the pancreatic cancer Participant User File. Thirty- and 90-day mortality rates and survival rates were determined for patients undergoing PD for primary tumor resection and compared with patients who had no surgery or metastasectomy. The Kaplan-Meier method was used to compare survival time. Cox regression models were used to assess factors independently associated with overall survival time. RESULTS: Resection of the primary tumor or metastatic disease each significantly improved overall survival time compared with no resection. Adding metastasectomy to PD resulted in an incremental increase in overall survival time. Both PD and metastasectomy are independently associated with overall survival time. CONCLUSIONS: Our report highlights the potential for survival time benefit in appropriately selected patients who undergo PD in the setting of metastatic PNET.

Lymph node metastases in resected pancreatic ductal adenocarcinoma: predictors of disease recurrence and survival.

BACKGROUND: Few studies have simultaneously assessed the prognostic value of the multiple classification systems for lymph node (LN) metastases in resected pancreatic ductal adenocarcinoma (PDAC). METHODS: In 600 patients with resected PDAC, we examined the association of LN parameters (AJCC 7th and 8th editions, LN ratio (LNR), and log odds of metastatic LN (LODDS)) with pattern of recurrence and patient survival using logistic regression and Cox proportional hazards regression, respectively. Regression models adjusted for age, sex, margin status, tumour grade, and perioperative therapy. RESULTS: Lymph node metastases classified by AJCC 7th and 8th editions, LNR, and LODDS were associated with worse disease free-survival (DFS) and overall survival (OS) (all Ptrend<0.01). American Joint Committee on Cancer 8th edition effectively predicted DFS and OS, while minimising model complexity. Lymph node metastases had weaker prognostic value in patients with positive margins and distal resections (both Pinteraction<0.03). Lymph node metastases by AJCC 7th and 8th editions did not predict the likelihood of local disease as the first site of recurrence. CONCLUSIONS: American Joint Committee on Cancer 8th edition LN classification is an effective and practical tool to predict outcomes in patients with resected PDAC. However, the prognostic value of LN metastases is attenuated in patients with positive resection margins and distal pancreatectomies.

Management of Neuroendocrine Tumor Liver Metastases: Long-Term Outcomes and Prognostic Factors from a Large Prospective Database.

BACKGROUND: Liver-directed therapies have been used to treat neuroendocrine liver metastases (NELM) for both symptomatic improvement and tumor growth control. We reviewed our experience with NELM to investigate the outcomes of available treatment modalities and to identify prognostic factors for survival. METHODS: We identified all patients with NELM, who were managed at our institution, from a prospectively collected institutional database. Overall survival (OS) was determined for each treatment modality. RESULTS: Between 2003 and 2010, we identified 939 patients with neuroendocrine tumors, of whom 649 patients had NELM. The primary tumor site was the small intestine in 245 patients (38%) and pancreas in 194 patients (30%). With a median follow-up of 44 months, the median, 5 and 10 year OS for each treatment group was as follows: hepatic resection (n = 58, 9%), 160 months, 90%, 70%; radiofrequency ablation (n = 28, 4%), 123 months, 84%, 55%; chemoembolization (n = 130, 20%), 66 months, 55%, 28%; systemic therapy (n = 316, 49%), 70 months, 58%, 31%; and observation (n = 117, 18%), 38 months, 38%, 20%. Age [hazard ratio (HR) 1.0, p < 0.001), small bowel primary site (HR 0.5, p < 0.001), hepatic resection (HR 0.3, p = 0.001), well-differentiated tumors (HR 0.3, p < 0.001), alkaline phosphatase within normal limit (WNL) (HR 0.4, p < 0.001), and chromogranin A WNL (HR 0.5, p < 0.001) were significant independent prognosticators for OS. CONCLUSIONS: This series represents one of the largest single-institution studies of NELM reported. We found that hepatic resection was associated with highly favorable OS. Our observations support hepatic resection in appropriately selected patients.

Saturation Mutagenesis of the Antithrombin Reactive Center Loop P14 Residue Supports a Three-step Mechanism of Heparin Allosteric Activation Involving Intermediate and Fully Activated States.

Past studies have suggested that a key feature of the mechanism of heparin allosteric activation of the anticoagulant serpin, antithrombin, is the release of the reactive center loop P14 residue from a native state stabilizing interaction with the hydrophobic core. However, more recent studies have indicated that this structural change plays a secondary role in the activation mechanism. To clarify this role, we expressed and characterized 15 antithrombin P14 variants. The variants exhibited basal reactivities with factors Xa and IXa, heparin affinities and thermal stabilities that were dramatically altered from wild type, consistent with the P14 mutations perturbing native state stability and shifting an allosteric equilibrium between native and activated states. Rapid kinetic studies confirmed that limiting rate constants for heparin allosteric activation of the mutants were altered in conjunction with the observed shifts of the allosteric equilibrium. However, correlations of the P14 mutations' effects on parameters reflecting the allosteric activation state of the serpin were inconsistent with a two-state model of allosteric activation and suggested multiple activated states. Together, these findings support a minimal three-state model of allosteric activation in which the P14 mutations perturb equilibria involving distinct native, intermediate, and fully activated states wherein the P14 residue retains an interaction with the hydrophobic core in the intermediate state but is released from the core in the fully activated state, and the bulk of allosteric activation has occurred in the intermediate.

Neoadjuvant irinotecan, cisplatin, and concurrent radiation therapy with celecoxib for patients with locally advanced esophageal cancer.

BACKGROUND: Patients with locally advanced esophageal cancer who are treated with trimodality therapy have a high recurrence rate. Preclinical evidence suggests that inhibition of cyclooxygenase 2 (COX2) increases the effectiveness of chemoradiation, and observational studies in humans suggest that COX-2 inhibition may reduce esophageal cancer risk. This trial tested the safety and efficacy of combining a COX2 inhibitor, celecoxib, with neoadjuvant irinotecan/cisplatin chemoradiation. METHODS: This single arm phase 2 trial combined irinotecan, cisplatin, and celecoxib with concurrent radiation therapy. Patients with stage IIA-IVA esophageal cancer received weekly cisplatin 30 mg/m(2) plus irinotecan 65 mg/m(2) on weeks 1, 2, 4, and 5 concurrently with 5040 cGy of radiation therapy. Celecoxib 400 mg was taken orally twice daily during chemoradiation, up to 1 week before surgery, and for 6 months following surgery. RESULTS: Forty patients were enrolled with stage IIa (30 %), stage IIb (20 %), stage III (22.5 %), and stage IVA (27.5 %) esophageal or gastroesophageal junction cancer (AJCC, 5th Edition). During chemoradiation, grade 3-4 treatment-related toxicity included dysphagia (20 %), anorexia (17.5 %), dehydration (17.5 %), nausea (15 %), neutropenia (12.5 %), diarrhea (10 %), fatigue (7.5 %), and febrile neutropenia (7.5 %). The pathological complete response rate was 32.5 %. The median progression free survival was 15.7 months and the median overall survival was 34.7 months. 15 % (n = 6) of patients treated on this study developed brain metastases. CONCLUSIONS: The addition of celecoxib to neoadjuvant cisplatin-irinotecan chemoradiation was tolerable; however, overall survival appeared comparable to prior studies using neoadjuvant cisplatin-irinotecan chemoradiation alone. Further studies adding celecoxib to neoadjuvant chemoradiation in esophageal cancer are not warranted. TRIAL REGISTRATION: Clinicaltrials.gov: NCT00137852 , registered August 29, 2005.

A standardized method for endoscopic necrosectomy improves complication and mortality rates.

OBJECTIVES: Endoscopic necrosectomy is effective in the treatment of walled-off necrosis (WON), and is preferred to surgical approaches, however complication and mortality rates remain high with few centers regularly employing the technique. Lack of a standardized approach may also contribute to these limitations. METHODS: Prior to the study, a multidisciplinary team applied standardized care assessment and management plan principles to develop and optimize a systematic approach for the management of WON. Preoperative, postoperative, and endoscopic management were standardized. Patient preparation, room set-up, technical features (EUS-guidance, cold-access with balloon dilation, fragmentation of necrosis on the initial procedure, antibiotic lavage, double pigtail stents), and discontinuation of PPIs to encourage auto-digestion of necrosis were included. This study employed a consecutive prospective clinical registry to assess the clinical outcomes of this standardized approach. RESULTS: 60 consecutive patients underwent 1.58 ± 0.1 necrosectomies, with debridement accomplished on the initial procedure in 98.3%. 39 patients (65%) required only one session. Clinical resolution occurred in 86.7%, with radiologic confirmation. Percutaneous drainage was required in 8 patients during follow-up, and 4 of these later required surgery. Serious adverse events occurred in 3.3% of patients, and there was no mortality. CONCLUSIONS: The standardized technique employed in this series was associated with lower rates of adverse events, morbidity, and mortality than prior large series. Use of a systematic approach, and integrating elements of this method may improve the risk profile of endoscopic necrosectomy and allow broader adoption.

Conformational activation of antithrombin by heparin involves an altered exosite interaction with protease.

Heparin allosterically activates antithrombin as an inhibitor of factors Xa and IXa by enhancing the initial Michaelis complex interaction of inhibitor with protease through exosites. Here, we investigate the mechanism of this enhancement by analyzing the effects of alanine mutations of six putative antithrombin exosite residues and three complementary protease exosite residues on antithrombin reactivity with these proteases in unactivated and heparin-activated states. Mutations of antithrombin Tyr(253) and His(319) exosite residues produced massive 10-200-fold losses in reactivity with factors Xa and IXa in both unactivated and heparin-activated states, indicating that these residues made critical attractive interactions with protease independent of heparin activation. By contrast, mutations of Asn(233), Arg(235), Glu(237), and Glu(255) exosite residues showed that these residues made both repulsive and attractive interactions with protease that depended on the activation state and whether the critical Tyr(253)/His(319) residues were mutated. Mutation of factor Xa Arg(143), Lys(148), and Arg(150) residues that interact with the exosite in the x-ray structure of the Michaelis complex confirmed the importance of all residues for heparin-activated antithrombin reactivity and Arg(150) for native serpin reactivity. These results demonstrate that the exosite is a key determinant of antithrombin reactivity with factors Xa and IXa in the native as well as the heparin-activated state and support a new model of allosteric activation we recently proposed in which a balance between attractive and repulsive exosite interactions in the native state is shifted to favor the attractive interactions in the activated state through core conformational changes induced by heparin binding.

The protease cathepsin L regulates Th17 cell differentiation.

Previously we reported that IL-17(+) T cells, primarily IL-17(+) γδ cells, are increased in mice lacking the protease inhibitor serpinB1 (serpinb1(-/-) mice). Here we show that serpinB1-deficient CD4 cells exhibit a cell-autonomous and selective deficiency in suppressing T helper 17 (Th17) cell differentiation. This suggested an opposing role for one or more protease in promoting Th17 differentiation. We found that several SerpinB1-inhibitable cysteine cathepsins are induced in Th17 cells, most prominently cathepsin L (catL); this was verified by peptidase assays, active site labeling and Western blots. Moreover, Th17 differentiation was suppressed by both broad cathepsin inhibitors and catL selective inhibitors. CatL is present in Th17 cells as single chain (SC)- and two-chain (TC)-forms. Inhibiting asparagine endopeptidase (AEP) blocked conversion of SC-catL to TC-catL and increased generation of serpinb1(-/-) Th17 cells, but not wild-type Th17 cells. These findings suggest that SC-catL is biologically active in promoting Th17 generation and is counter-regulated by serpinB1 and secondarily by AEP. Thus, in addition to regulation by cytokines and transcription factors, differentiation of CD4 cells to Th17 cells is actively regulated by a catL-serpinB1-AEP module. Targeting this protease regulatory module could be an approach to treating Th17 cell-driven autoimmune disorders.

Kinetic intermediates en route to the final serpin-protease complex: studies of complexes of α1-protease inhibitor with trypsin.

Serpin protein protease inhibitors inactivate their target proteases through a unique mechanism in which a major serpin conformational change, resulting in a 70-Å translocation of the protease from its initial reactive center loop docking site to the opposite pole of the serpin, kinetically traps the acyl-intermediate complex. Although the initial Michaelis and final trapped acyl-intermediate complexes have been well characterized structurally, the intermediate stages involved in this remarkable transformation are not well understood. To better characterize such intermediate steps, we undertook rapid kinetic studies of the FRET and fluorescence perturbation changes of site-specific fluorophore-labeled derivatives of the serpin, α1-protease inhibitor (α1PI), which report the serpin and protease conformational changes involved in transforming the Michaelis complex to the trapped acyl-intermediate complex in reactions with trypsin. Two kinetically resolvable conformational changes were observed in the reactions, ascribable to (i) serpin reactive center loop insertion into sheet A with full protease translocation but incomplete protease distortion followed by, (ii) full conformational distortion and movement of the protease and coupled serpin conformational changes involving the F helix-sheet A interface. Kinetic studies of calcium effects on the labeled α1PI-trypsin reactions demonstrated both inactive and low activity states of the distorted protease in the final complex that were distinct from the intermediate distorted state. These studies provide new insights into the nature of the serpin and protease conformational changes involved in trapping the acyl-intermediate complex in serpin-protease reactions and support a previously proposed role for helix F in the trapping mechanism.

The allosteric mechanism of activation of antithrombin as an inhibitor of factor IXa and factor Xa: heparin-independent full activation through mutations adjacent to helix D.

Allosteric conformational changes in antithrombin induced by binding a specific heparin pentasaccharide result in very large increases in the rates of inhibition of factors IXa and Xa but not of thrombin. These are accompanied by CD, fluorescence, and NMR spectroscopic changes. X-ray structures show that heparin binding results in extension of helix D in the region 131-136 with coincident, and possibly coupled, expulsion of the hinge of the reactive center loop. To examine the importance of helix D extension, we have introduced strong helix-promoting mutations in the 131-136 region of antithrombin (YRKAQK to LEEAAE). The resulting variant has endogenous fluorescence indistinguishable from WT antithrombin yet, in the absence of heparin, shows massive enhancements in rates of inhibition of factors IXa and Xa (114- and 110-fold, respectively), but not of thrombin, together with changes in near- and far-UV CD and (1)H NMR spectra. Heparin binding gives only ∼3-4-fold further rate enhancement but increases tryptophan fluorescence by ∼23% without major additional CD or NMR changes. Variants with subsets of these mutations show intermediate activation in the absence of heparin, again with basal fluorescence similar to WT and large increases upon heparin binding. These findings suggest that in WT antithrombin there are two major complementary sources of conformational activation of antithrombin, probably involving altered contacts of side chains of Tyr-131 and Ala-134 with core hydrophobic residues, whereas the reactive center loop hinge expulsion plays only a minor additional role.

The 90-day mortality after pancreatectomy for cancer is double the 30-day mortality: more than 20,000 resections from the national cancer data base.

BACKGROUND: Operative mortality traditionally has been defined as the rate within 30 days or during the initial hospitalization, and studies that established the volume-outcome relationship for pancreatectomy used similar definitions. METHODS: Pancreatectomies reported to the National Cancer Data Base (NCDB) during 2007-2010 were examined for 30- and 90-day mortality. Unadjusted mortality rates were compared by type of resection, stage, comorbidities, and average annual hospital volume. Hierarchical logistic regression models generated risk-adjusted odds ratios for 30- and 90-day mortality. RESULTS: After 21,482 pancreatectomies, the unadjusted 30-day mortality rate was 3.7 % (95 % confidence interval [CI] 3.4-3.9 %), which doubled at 90 days to 7.4 % (95 % CI 7.0-7.8). The unadjusted and risk-adjusted mortality rates were higher at 30 days with increasing age, increasing stage, male gender, lower income, low hospital volume, resections other than distal pancreatectomy, Medicare or Medicaid insurance coverage, residence in a Southern census division, history of prior cancer, and multiple comorbidities. The lowest-volume hospitals (<5 per year) performed 19 % of the pancreatectomies, with a risk-adjusted odds ratios for mortality that were 4.2 times higher (95 % CI 3.1-5.8) at 30 days and remained 1.9 times higher (95 % CI 1.5-2.3) at 30-90 days compared with hospitals that had high volumes (≥40 per year). CONCLUSION: Mortality rates within 90 days after pancreatic resection are double those at 30 days. The volume-outcome relationship persists in the NCDB. Reporting mortality rates 90 days after pancreatectomy is important. Hospitals should be aware of their annual volume and mortality rates 30 and 90 days after pancreatectomy and should benchmark the use of high-volume hospitals.

Specificity and selectivity profile of EP217609: a new neutralizable dual-action anticoagulant that targets thrombin and factor Xa.

EP217609 is a new dual-action parenteral anticoagulant that combines an indirect factor Xa inhibitor (fondaparinux analog) and a direct thrombin inhibitor (α-NAPAP analog) in a single molecule together with a biotin tag to allow avidin neutralization. EP217609 exhibits an unprecedented pharmacologic profile in showing high bioavailability, long plasma half-life, and potent antithrombotic activity in animals without the complications of thrombin rebound. Here we report the exceptional specificity and selectivity profile of EP217609. EP217609 inhibited thrombin with rapid kinetics (k(on) > 10(7)M(-1)s(-1)), a high affinity (K(I) = 30-40pM), and more than 1000-fold selectivity over other coagulation and fibrinolytic protease targets, comparing favorably with the best direct thrombin inhibitors known. EP217609 bound antithrombin with high affinity (K(D) = 30nM) and activated the serpin to rapidly (k(ass) ∼ 10(6)M(-1)s(-1)) and selectively (> 20-fold) inhibit factor Xa. The dual inhibitor moieties of EP217609 acted largely independently with only modest linkage effects of ligand occupancy of one inhibitor moiety on the potency of the other (∼ 5-fold). In contrast, avidin binding effectively neutralized the potency of both inhibitor moieties (20- to 100-fold). These findings demonstrate the superior anticoagulant efficacy and rapid avidin neutralizability of EP217609 compared with anticoagulants that target thrombin or factor Xa alone.

Phase II trial of vaccination with autologous, irradiated melanoma cells engineered by adenoviral mediated gene transfer to secrete granulocyte-macrophage colony stimulating factor in patients with stage III and IV melanoma.

Background: In the era of immune checkpoint blockade, the role of cancer vaccines in immune priming has provided additional potential for therapeutic improvements. Prior studies have demonstrated delayed type hypersensitivity and anti-tumor immunity with vaccines engineered to secrete granulocyte-macrophage colony-stimulating factor (GM-CSF). The safety, efficacy and anti-tumor immunity of GM-CSF secreting vaccine in patients with previously treated stage III or IV melanoma needs further investigation. Methods: In this phase II trial, excised lymph node metastases were processed to single cells, transduced with an adenoviral vector encoding GM-CSF, irradiated, and cryopreserved. Individual vaccines were composed of 1x106, 4x106, or 1x107 tumor cells, and were injected intradermally and subcutaneously at weekly and biweekly intervals. The primary endpoints were feasibility of producing vaccine in stage III patients and determining the proportion of patients alive at two years in stage IV patients. Results: GM-CSF vaccine was successfully developed and administered in all 61 patients. Toxicities were restricted to grade 1-2 local skin reactions. The median OS for stage III patients (n = 20) was 71.1 (95% CI, 43.7 to NR) months and 14.9 (95%CI, 12.1 to 39.7) months for stage IV patients. The median PFS in stage III patients was 50.7 (95%CI, 36.3 to NR) months and 4.1 (95% CI, 3.0-6.3) months in stage IV patients. In the overall population, the disease control rate was 39.3% (95%CI, 27.1 to 52.7%). In stage III patients, higher pre-treatment plasma cytokine levels of MMP-1, TRAIL, CXCL-11, CXCL-13 were associated with improved PFS (p<0.05 for all). An increase in post-vaccination levels of IL-15 and TRAIL for stage III patients was associated with improved PFS (p=0.03 for both). Similarly, an increase in post-vaccination IL-16 level for stage IV patients was associated with improved PFS (p=0.02) and clinical benefit. Conclusions: Vaccination with autologous melanoma cells secreting GM-CSF augments antitumor immunity in stage III and IV patients with melanoma, is safe, and demonstrates disease control. Luminex data suggests that changes in inflammatory cytokines and immune cell infiltration promote tumor antigen presentation and subsequent tumor cell destruction. Additional investigation to administer this vaccine in combination with immune checkpoint inhibitors is needed.

The minimum work requirements for atmospheric water harvesting.

Idealized cycles for the three most common methods of atmospheric water harvesting, membrane, desiccant, and condenser, are analyzed. It is found that they all have substantially the same efficiency as a function of water removal fraction. In addition, for small removal fractions they all approach the minimum thermodynamic work requirement. This minimum is shown to come from the entropy of mixing at the water-atmosphere boundary. For larger removal fractions, additional work is required, which is shown to come from mixing the drier output air with ambient air.

Protecting patients from an unsafe system: the etiology and recovery of intraoperative deviations in care.

OBJECTIVE: To understand the etiology and resolution of unanticipated events in the operating room (OR). BACKGROUND: The majority of surgical adverse events occur intraoperatively. The OR represents a complex, high-risk system. The influence of different human, team, and organizational/environmental factors on safety and performance is unknown. METHODS: We video-recorded and transcribed 10 high-acuity operations, representing 43.7 hours of patient care. Deviations, defined as delays and/or episodes of decreased patient safety, were identified by majority consensus of a multidisciplinary team. Factors that contributed to each event and/or mitigated its impact were determined and attributed to the patient, providers, or environment/organization. RESULTS: Thirty-three deviations (10 delays, 17 safety compromises, 6 both) occurred--with a mean of 1 every 79.4 minutes. These deviations were multifactorial (mean 3.1 factors). Problems with communication and organizational structure appeared repeatedly at the root of both types of deviations. Delays tended to be resolved with vigilance, communication, coordination, and cooperation, while mediation of safety compromises was most frequently accomplished with vigilance, leadership, communication, and/or coordination. The organization/environment was not found to play a direct role in compensation. CONCLUSIONS: Unanticipated events are common in the OR. Deviations result from poor organizational/environmental design and suboptimal team dynamics, with caregivers compensating to avoid patient harm. Although recognized in other high-risk domains, such human resilience has not yet been described in surgery and has major implications for the design of safety interventions.

Endoscopic pyloric balloon dilatation obviates the need for pyloroplasty at esophagectomy.

BACKGROUND: Because the rate of acquired pyloric stenosis (APS) from truncal vagotomy is 15%, many surgeons perform pyloroplasty or pyloromyotomy at the time of esophagectomy. Endoscopic pyloric balloon dilatation (EPBD) is another method to manage APS. This study evaluated a cohort treated with preoperative EPBD. METHODS: This is a retrospective review of all patients treated with preoperative EPBD and esophagectomy for cancer from 2002 to 2009 at Brigham and Women's Hospital, a tertiary care center. Outcome measures included need for subsequent surgery for gastric outlet obstruction, rate of pyloric stenosis noted on postoperative endoscopy, and complications. RESULTS: Upon review of the series, 25 patients (80% male; median age, 63 [range 47-81] years) had outpatient preoperative EPBD and esophagectomies 1-2 weeks later and were included in the study. None had pyloroplasties or pyloromyotomies at the time of esophagectomy. Selected patients had postoperative endoscopy. Of the 25 patients, 20 had transhiatal esophagectomies, 3 had thoracoabdominal esophagectomies, and 2 had VATS 3-hole esophagectomies. Median follow-up time was 22 (range, 1-84) months. There were no complications from EPBD. There were no postoperative deaths. No patient needed a second operation for gastric outlet obstruction. All patients had postoperative barium swallows (BaS) or endoscopy or both. Only one patient (4%) required one postoperative EPBD to dilate a 16-mm pylorus. Three others had delayed gastric emptying on BaS with endoscopy showing each pylorus was wide open. Their symptoms improved with time. CONCLUSIONS: In this cohort, preoperative EPBD in all patients combined with postoperative EPBD in one patient obviated the need for pyloroplasty. This approach merits further study in a larger cohort, particularly to determine whether preoperative EPBD is necessary or if only selected postoperative EPBD is sufficient.