Serine protease inhibitor, SerpinA3n, regulates cardiac remodelling after myocardial infarction.

AIMS: Following myocardial infarction (MI), the heart repairs itself via a fibrotic repair response. The degree of fibrosis is determined by the balance between deposition of extracellular matrix (ECM) by activated fibroblasts and breakdown of nascent scar tissue by proteases that are secreted predominantly by inflammatory cells. Excessive proteolytic activity and matrix turnover has been observed in human heart failure, and protease inhibitors in the injured heart regulate matrix breakdown. Serine protease inhibitors (Serpins) represent the largest and the most functionally diverse family of evolutionary conserved protease inhibitors, and levels of the specific Serpin, SerpinA3, have been strongly associated with clinical outcomes in human MI as well as non-ischaemic cardiomyopathies. Yet, the role of Serpins in regulating cardiac remodelling is poorly understood. The aim of this study was to understand the role of Serpins in regulating scar formation after MI. METHODS AND RESULTS: Using a SerpinA3n conditional knockout mice model, we observed the robust expression of Serpins in the infarcted murine heart and demonstrate that genetic deletion of SerpinA3n (mouse homologue of SerpinA3) leads to increased activity of substrate proteases, poorly compacted matrix, and significantly worse post-infarct cardiac function. Single-cell transcriptomics complemented with histology in SerpinA3n-deficient animals demonstrated increased inflammation, adverse myocyte hypertrophy, and expression of pro-hypertrophic genes. Proteomic analysis of scar tissue demonstrated decreased cross-linking of ECM peptides consistent with increased proteolysis in SerpinA3n-deficient animals. CONCLUSION: Our study demonstrates a hitherto unappreciated causal role of Serpins in regulating matrix function and post-infarct cardiac remodelling.

Assessing Target Specificity of the Small Molecule Inhibitor MARIMASTAT to Snake Venom Toxins: A Novel Application of Thermal Proteome Profiling.

New treatments that circumvent the pitfalls of traditional antivenom therapies are critical to address the problem of snakebite globally. Numerous snake venom toxin inhibitors have shown promising cross-species neutralization of medically significant venom toxins in vivo and in vitro. The development of high-throughput approaches for the screening of such inhibitors could accelerate their identification, testing, and implementation and thus holds exciting potential for improving the treatments and outcomes of snakebite envenomation worldwide. Energetics-based proteomic approaches, including thermal proteome profiling and proteome integral solubility alteration (PISA) assays, represent "deep proteomics" methods for high throughput, proteome-wide identification of drug targets and ligands. In the following study, we apply thermal proteome profiling and PISA methods to characterize the interactions between venom toxin proteoforms in Crotalus atrox (Western Diamondback Rattlesnake) and the snake venom metalloprotease (SVMP) inhibitor marimastat. We investigate its venom proteome-wide effects and characterize its interactions with specific SVMP proteoforms, as well as its potential targeting of non-SVMP venom toxin families. We also compare the performance of PISA thermal window and soluble supernatant with insoluble precipitate using two inhibitor concentrations, providing the first demonstration of the utility of a sensitive high-throughput PISA-based approach to assess the direct targets of small molecule inhibitors for snake venom.

Sensory nerve release of CGRP increases tumor growth in HNSCC by suppressing TILs.

BACKGROUND: Perineural invasion (PNI) and nerve density within the tumor microenvironment (TME) have long been associated with worse outcomes in head and neck squamous cell carcinoma (HNSCC). This prompted an investigation into how nerves within the tumor microenvironment affect the adaptive immune system and tumor growth. METHODS: We used RNA sequencing analysis of human tumor tissue from a recent HNSCC clinical trial, proteomics of human nerves from HNSCC patients, and syngeneic orthotopic murine models of HPV-unrelated HNSCC to investigate how sensory nerves modulate the adaptive immune system. FINDINGS: Calcitonin gene-related peptide (CGRP) directly inhibited CD8 T cell activity in vitro, and blocking sensory nerve function surgically, pharmacologically, or genetically increased CD8 and CD4 T cell activity in vivo. CONCLUSIONS: Our data support sensory nerves playing a role in accelerating tumor growth by directly acting on the adaptive immune system to decrease Th1 CD4 T cells and activated CD8 T cells in the TME. These data support further investigation into the role of sensory nerves in the TME of HNSCC and points toward the possible treatment efficacy of blocking sensory nerve function or specifically inhibiting CGRP release or activity within the TME to improve outcomes. FUNDING: 1R01DE028282-01, 1R01DE028529-01, 1P50CA261605-01 (to S.D.K.), 1R01CA284651-01 (to S.D.K.), and F31 DE029997 (to L.B.D.).

Benefit of Surgical Resection of the Primary Tumor in Patients Undergoing Chemotherapy for Stage IV Colorectal Cancer with Unresected Metastasis.

PURPOSE: Resection of the primary tumor in patients with unresected metastatic colorectal cancer is controversial, and often performed only for palliation of symptoms. Our goal was to determine if resection of the primary tumor in this patient population is associated with improved survival. METHODS: This is a retrospective cohort study of the National Cancer Data Base from 2004 to 2012. The study population included all patients with synchronous metastatic colorectal adenocarcinoma who were treated with systemic chemotherapy. The study groups were patients who underwent definitive surgery for the primary tumor and those who did not. Patients were excluded if they had surgical intervention on the sites of metastasis or pathology other than adenocarcinoma. Primary outcome was overall survival. RESULTS: Of the 65,543 patients with unresected stage IV colorectal adenocarcinoma undergoing chemotherapy, 55% underwent surgical resection of the primary site. Patients who underwent surgical resection of the primary tumor had improved median survival compared to patients treated with chemotherapy alone (22 vs 13 months, p < .0001). The surgical survival benefit was present for patients who were treated with either multi-agent or single-agent chemotherapy (23 vs 14 months, p < 0.001; 19 vs 9 months, p < 0.001). Surgical resection of the primary tumor was also associated with improved survival when using multivariate analysis with propensity score matching (OR = 0.863; 95% CI [0.805-.924]; HR = 0.914; 95% CI [0.888-0.942]). CONCLUSIONS: Our results show that in patients with synchronous unresected stage IV colorectal adenocarcinoma undergoing single- or multi-agent chemotherapy, after adjusting for confounding variables, definitive resection of the primary site was associated with improved overall survival. Large randomized controlled trials are needed to determine if there is a causal relationship between surgery and increased overall survival in this patient population.

Complications of Diverting Ileostomy after Low Anterior Resection for Rectal Carcinoma.

There have been few studies directly comparing the postoperative complications in patients with a diverting ileostomy to patients who were not diverted after low anterior resection (LAR) for rectal carcinoma. This study is a retrospective chart review of all rectal carcinoma patients (99) who underwent a LAR from January 2009 to December 2014 at Loma Linda University Medical Center and Veterans Affairs Loma Linda Healthcare System. A majority of patients were diverted (58% vs 42%). The diverted patients were more likely to have a low tumor location (P < 0.01), preoperative chemoradiation (P < 0.01), and more intraoperative blood loss (P < 0.01). Our study shows a statistically significant higher overall complication rate among patients receiving a diverting ileostomy in the six months after LAR (61% vs 38%, P = 0.02). The difference is due to a higher rate of readmission (27% vs 14%) and acute kidney injury (14% vs 5%) in patients with a diverting ileostomy. It also shows that there is a higher rate of unplanned reoperation (11% vs 6%) due to anastomotic leak (17% vs 5%) in nondiverted patients. Further studies are needed to refine the specific indications to maximize the benefit of diverting ileostomy after LAR for rectal carcinoma.

The role of time and pressure on alveolar recruitment.

Inappropriate mechanical ventilation in patients with acute respiratory distress syndrome can lead to ventilator-induced lung injury (VILI) and increase the morbidity and mortality. Reopening collapsed lung units may significantly reduce VILI, but the mechanisms governing lung recruitment are unclear. We thus investigated the dynamics of lung recruitment at the alveolar level. Rats (n = 6) were anesthetized and mechanically ventilated. The lungs were then lavaged with saline to simulate acute respiratory distress syndrome (ARDS). A left thoracotomy was performed, and an in vivo microscope was placed on the lung surface. The lung was recruited to three recruitment pressures (RP) of 20, 30, or 40 cmH(2)O for 40 s while subpleural alveoli were continuously filmed. Following measurement of microscopic alveolar recruitment, the lungs were excised, and macroscopic gross lung recruitment was digitally filmed. Recruitment was quantified by computer image analysis, and data were interpreted using a mathematical model. The majority of alveolar recruitment (78.3 +/- 7.4 and 84.6 +/- 5.1%) occurred in the first 2 s (T2) following application of RP 30 and 40, respectively. Only 51.9 +/- 5.4% of the microscopic field was recruited by T2 with RP 20. There was limited recruitment from T2 to T40 at all RPs. The majority of gross lung recruitment also occurred by T2 with gradual recruitment to T40. The data were accurately predicted by a mathematical model incorporating the effects of both pressure and time. Alveolar recruitment is determined by the magnitude of recruiting pressure and length of time pressure is applied, a concept supported by our mathematical model. Such a temporal dependence of alveolar recruitment needs to be considered when recruitment maneuvers for clinical application are designed.