CaMKIV-dependent preservation of mTOR expression is required for autophagy during lipopolysaccharide-induced inflammation and acute kidney injury.

Autophagy, an evolutionarily conserved homeostasis process regulating biomass quantity and quality, plays a critical role in the host response to sepsis. Recent studies show its calcium dependence, but the calcium-sensitive regulatory cascades have not been defined. In this study, we describe a novel mechanism in which calcium/calmodulin-dependent protein kinase IV (CaMKIV), through inhibitory serine phosphorylation of GSK-3ß and inhibition of FBXW7 recruitment, prevents ubiquitin proteosomal degradation of mammalian target of rapamycin (mTOR) and thereby augments autophagy in both the macrophage and the kidney. Under the conditions of sepsis studied, mTOR expression and activity were requisite for autophagy, a paradigm countering the current perspective that prototypically, mTOR inhibition induces autophagy. CaMKIV-mTOR-dependent autophagy was fundamentally important for IL-6 production in vitro and in vivo. Similar mechanisms were operant in the kidney during endotoxemia and served a cytoprotective role in mitigating acute kidney injury. Thus, CaMKIV-mTOR-dependent autophagy is conserved in both immune and nonimmune/parenchymal cells and is fundamental for the respective functional and adaptive responses to septic insult.

Adrenal Imaging Features Predict Malignancy Better than Tumor Size.

INTRODUCTION: In adrenal tumors, size ≥ 4 cm has been an indication for adrenalectomy due to concern for malignancy. We compared mass size to imaging features (ImF) for accuracy in diagnosing adrenal malignancy. METHODS: Data were retrieved for 112 consecutive patients who had adrenalectomy from January 2011 to August 2014. ImF was classified as nonbenign if HU > 10 on unenhanced CT scan or if loss of signal on out-of-phase imaging was absent on chemical-shift MRI. Indications for resection included hormonal hypersecretion, nonbenign ImF, and/or size ≥ 4 cm. RESULTS: Of 113 resected adrenals, 37 % were functional. Histologic malignancy occurred in 18 % (20/113) and included 3 adrenocortical carcinomas (ACC), 1 epithelioid liposarcoma, 1 lymphoma, 1 malignant nerve sheath tumor, and 14 adrenal metastases. Patients with malignancies were older (mean age, 60 ± 13 vs. 51 ± 14 years, p = 0.01). Malignant tumors were larger on preoperative imaging (mean 5.3 ± 3.2 vs. 3.9 ± 2.4 cm, p = 0.03). All 20 malignant masses had nonbenign ImF. In predicting malignancy, the sensitivity, specificity, NPV, and PPV of nonbenign ImF was 100, 57, 100, and 33 %, respectively. Size ≥ 4 cm was less predictive with sensitivity, specificity, NPV, and PPV of 55, 61, 86, and 23 %, respectively. If size ≥ 4 cm had been used as the sole criterion for surgery, 45 % of malignancies (9/20) would have been missed including 8 metastases and an ACC. CONCLUSIONS: In resected adrenal tumors, the presence of nonbenign ImF is more sensitive for malignancy than mass size (100 vs. 55 %) with equivalent specificity. Regardless of mass size, adrenalectomy should be strongly considered when non-benign ImF are present.

CaMKIα regulates AMP kinase-dependent, TORC-1-independent autophagy during lipopolysaccharide-induced acute lung neutrophilic inflammation.

Autophagy is an evolutionarily conserved cytoplasmic process regulated by the energy rheostats mammalian target of rapamycin and AMP kinase (AMPK) that recycles damaged or unused proteins and organelles. It has been described as an important effector arm of immune cells. We have shown that the cytoplasmically oriented calcium/calmodulin-dependent protein kinase (CaMK)Iα regulates the inflammatory phenotype of the macrophage (M). In this study, we hypothesize that CaMKIα mediates M autophagy. LPS induced autophagy in RAW 264.7 cells and murine peritoneal M that was attenuated with biochemical CaMK inhibition or CaMKIα small interfering RNA (siRNA). Inhibition of CaMKIα reduced LPS-induced p-Thr(172)AMPK and target of rapamycin complex-1 activity, and expression of a constitutively active CaMKIα but not a kinase-deficient mutant induced p-Thr(172)AMPK and autophagy that was attenuated by the AMPK inhibitor compound C. Coimmunoprecipitation and in vitro kinase assays demonstrated that CaMKIα activates AMPK, thereby inducing ATG7, which also localizes to this CaMKIα/AMPK complex. During LPS-induced lung inflammation, C57BL/6 mice receiving CaMKIα(siRNA) displayed reduced lung and bronchoalveolar immune cell autophagy that correlated with reduced neutrophil recruitment, myeloperoxidase activity, and air space cytokine concentration. Independently inhibiting autophagy, using siRNA targeting the PI3K VPS34, yielded similar reductions in lung autophagy and neutrophil recruitment. Thus, a novel CaMKIα/AMPK pathway is rapidly activated in M exposed to LPS and regulates an early autophagic response, independent of target of rapamycin complex-1 inhibition. These mechanisms appear to be operant in vivo in orchestrating LPS-induced lung neutrophil recruitment and inflammation.

The small abnormal parathyroid gland is increasingly common and heralds operative complexity.

BACKGROUND: Over decades, improvements in presymptomatic screening and awareness of surgical benefits have changed the presentation and management of primary hyperparathyroidism (PHPT). Unrecognized multiglandular disease (MGD) remains a major cause of operative failure. We hypothesized that during parathyroid surgery the initial finding of a mildly enlarged gland is now frequent and predicts both MGD and failure. METHODS: A prospective database was queried to examine the outcomes of initial exploration for sporadic PHPT using intraoperative PTH monitoring (IOPTH) over 15 years. All patients had follow-up ≥6 months (mean = 1.8 years). Cure was defined by normocalcemia at 6 months and microadenoma by resected weight of <200 mg. RESULTS: Of the 1,150 patients, 98.9 % were cured and 15 % had MGD. The highest preoperative calcium level decreased over time (p < 0.001) and varied directly with adenoma weight (p < 0.001). Over time, single adenoma weight dropped by half (p = 0.002) and microadenoma was increasingly common (p < 0.01). MGD risk varied inversely with weight of first resected abnormal gland. Microadenoma required bilateral exploration more often than macroadenoma (48 vs. 18 %, p < 0.01). When at exploration the first resected gland was <200 mg, the rates of MGD (40 vs. 11 %, p = 0.001), inadequate initial IOPTH drop (67 vs. 79 %, p = 0.002), operative failure (6.6 vs. 0.7 %, p < 0.001), and long-term recurrence (1.6 vs. 0.3 %, p = 0.007) were higher. CONCLUSIONS: Single parathyroid adenomas are smaller than in the past and require more complex pre- and intraoperative management. During exploration for sporadic PHPT, a first abnormal gland <200 mg should heighten suspicion of MGD and presages a tenfold higher failure rate.

RAS mutations in thyroid cancer.

In recent years, our understanding of the genetic alterations underlying thyroid oncogenesis has greatly expanded. The use of molecular markers, including RAS, in the management of thyroid carcinoma is also increasing. This review summarizes the current literature surrounding RAS and discusses its potential as a diagnostic and prognostic indicator in the management of thyroid cancer.

Calcium supplementation during sepsis exacerbates organ failure and mortality via calcium/calmodulin-dependent protein kinase kinase signaling.

BACKGROUND: Calcium plays an essential role in nearly all cellular processes. As such, cellular and systemic calcium concentrations are tightly regulated. During sepsis, derangements in such tight regulation frequently occur, and treating hypocalcemia with parenteral calcium administration remains the current practice guideline. OBJECTIVE: We investigated whether calcium administration worsens mortality and organ dysfunction using an experimental murine model of sepsis and explored the mechanistic role of the family of calcium/calmodulin-dependent protein kinases in mediating these physiological effects. To highlight the biological relevance of these observations, we conducted a translational study of the association between calcium administration, organ dysfunction, and mortality among a cohort of critically ill septic ICU patients. DESIGN: Prospective, randomized controlled experimental murine study and observational clinical cohort analysis. SETTING: University research laboratory and eight ICUs at a tertiary care center. PATIENTS: A cohort of 870 septic ICU patients. SUBJECTS: C57Bl/6 and CaMKK mice. INTERVENTIONS: Mice underwent cecal ligation and puncture polymicrobial sepsis and were administered with calcium chloride (0.25 or 0.25 mg/kg) or normal saline. MEASUREMENTS AND MAIN RESULTS: Administering calcium chloride to septic C57Bl/6 mice heightened systemic inflammation and vascular leak, exacerbated hepatic and renal dysfunction, and increased mortality. These events were significantly attenuated in CaMKK mice. In a risk-adjusted analysis of septic patients, calcium administration was associated with an increased risk of death, odds ratio 1.92 (95% CI, 1.00-3.68; p = 0.049), a significant increase in the risk of renal dysfunction, odds ratio 4.74 (95% CI, 2.48-9.08; p < 0.001), and a significant reduction in ventilator-free days, mean decrease 3.29 days (0.50-6.08 days; p = 0.02). CONCLUSIONS: Derangements in calcium homeostasis occur during sepsis that is sensitive to calcium administration. This altered calcium signaling, transduced by the calmodulin-dependent protein kinase kinase cascade, mediates heightened inflammation and vascular leak that culminates in elevated organ dysfunction and mortality. In the clinical management of septic patients, calcium supplementation provides no benefit and may impose harm.

Outcome and prognostic factors after adrenalectomy for patients with distant adrenal metastasis.

BACKGROUND: The purpose of this study was to describe a single-institution experience with adrenal metastasectomy and to elucidate factors that may bear prognostic significance. METHODS: This is a single-center, retrospective review of patients with adrenal metastasis who underwent adrenalectomy performed with curative intent between 2000 and 2012. The Kaplan-Meier method was used to evaluate overall survival from time of adrenalectomy to death or last follow-up. Primary endpoint was death from any cause. Clinical variables were examined for association with survival. RESULTS: The study included 62 patients with mean age of 60 (±12) years; 55 % (34 of 62) were male, 85 % (53 of 62) presented with isolated adrenal metastasis, and 82 % (51 of 62) had metachronous disease with median disease-free interval (DFI) of 22 months (range, 6-217 months). Non-small cell lung cancer (NSCLC) was the most common primary comprising 50 % of cases. Median survival for the study population was 30 months (range, 1-145 months) and 5-year survival was 31 %. Patients with NSCLC had significantly shortened survival compared with non-NSCLC with median and 5-year survival of 17 versus 47 months and 27 % versus 38 %, respectively (p = .033). Synchronous metastasis (p = .028) and DFI < 12 months (p = .038) were also associated with worse survival outcome, though male gender (p = .69) and oligometastatic disease (p = .62) were not. CONCLUSIONS: Adrenal metastasectomy resulted in median survival of 30 months and 5-year survival of 31 %. Shorter survival was associated with lung primary, short disease-free interval, and synchronous metastasis, but not with the presence of oligometastatic disease provided that the primary cancer and additional metastatic lesions were adequately controlled and amenable to resection.

BRAF V600E mutation independently predicts central compartment lymph node metastasis in patients with papillary thyroid cancer.

PURPOSE: This study was designed to examine whether available preoperative clinical parameters, including B-type Raf kinase (BRAF) V600E mutation status, can identify patients at risk for central compartment lymph node metastasis (CLNM). METHODS: Under an institutional review board-approved protocol, we conducted a single-center, retrospective review of all patients who had initial thyroidectomy for histologic papillary thyroid carcinoma (PTC) during 2010. The presence of CLNM was examined for correlation with available preoperative clinical parameters, including tumor size, gender, age, and BRAF mutation status. RESULTS: Cervical lymph node resection and molecular testing on FNAB or histopathologic specimen was performed on a consecutive series of 156 study patients with histologic PTC. Overall, CLNM was diagnosed in 37% and 46% were BRAF-mutation-positive. BRAF positivity was the only clinical parameter associated with CLNM (BRAF, p=0.002; tumor size≥2 cm, p=0.16; male gender, p=0.1; age≥45 years, p=0.3) and remained an independent predictor of CLNM on multiple logistic regression analysis (odds ratio (OR) 3.2, p=0.001). The PPV and NPV of BRAF positivity for CLNM was 50 and 74%, respectively. When restricting the analysis to the subset of 38 patients who had molecular testing performed preoperatively on FNAB, the PPV and NPV of BRAF positivity for CLNM was 47 and 91%, respectively, and BRAF positivity was still a significant predictor of CLNM on both univariate (OR 8.4, p=0.01) and multivariate (OR 9.7, p=0.02) analyses. CONCLUSIONS: Of the commonly used clinical parameters available preoperatively, the BRAF V600E mutation is the only independent predictor of CLNM in PTC and can be utilized to guide the extent of initial surgery.

Both BRAF V600E mutation and older age (≥ 65 years) are associated with recurrent papillary thyroid cancer.

PURPOSE: This study was designed to examine the aggressive features of BRAF-positive papillary thyroid cancer (PTC) and association with age. METHODS: We compared the clinicopathologic parameters and BRAF V600E mutation status of 121 elderly (age ≥65 years) PTC patients who underwent thyroidectomy from January 2007 to December 2009 to a consecutive cohort of 98 younger (age <65 years) PTC patients. RESULTS: Younger and elderly PTC patients had similar incidences of BRAF-positive tumors (41% vs. 38%; p = 0.67). The elderly cohort was more likely to have smaller tumors (mean 1.6 vs. 2.1 cm; p = 0.001), present with advanced TNM stage (36% vs. 19%; p = 0.008), and have persistent/recurrent disease (10% vs. 1%; p = 0.006). BRAF-positive status was associated with PTC that were tall cell variant (p < 0.001), had extrathyroidal extension (p < 0.001), lymph node involvement (p = 0.008), advanced (III/IV) TNM stage (p < 0.001), and disease recurrence (p < 0.001). Except for lymph node involvement, the association between aggressive histology characteristics at presentation and BRAF-positive PTC also was observed within the age-defined cohorts. In short-term follow-up (mean, 18 months), persistent/recurrent PTC was much more likely to occur in patients who were both BRAF-positive and elderly (22%). CONCLUSIONS: BRAF mutations are equally present in younger and older patients. Aggressive histology characteristics at presentation are associated with BRAF-positive PTC, irrespective of age. However, the well-established association of BRAF with recurrence is limited to older (age ≥65 years) patients.

Delay to therapeutic interventional radiology postinjury: time is of the essence.

OBJECTIVE: Hemorrhage remains a leading cause of early death in injured patients, and definitive control of bleeding remains a fundamental principle of trauma management. Therapeutic interventional radiology (IR) procedures have increasingly become essential in the acute management of traumatic injury. The importance of time to control of hemorrhage for therapeutic IR procedures has not been adequately characterized. METHODS: A retrospective analysis was performed by using data derived from the National Trauma Data Bank, version 7.1. Inclusion criteria included the following: adult, hypotensive patients, scene admission, patients who underwent early therapeutic IR vascular occlusive procedures within in 3 hours of admission at a level I or II designated trauma center (n = 1,748). Exclusion criteria included intracranial or venous occlusion procedures, patients who underwent any abdominal, thoracic, vascular, or intracranial operative procedures throughout their entire hospital stay. Logistic regression analysis was used to analyze the independent mortality risk associated with DELAY to IR procedures after controlling for important confounders. RESULTS: The majority of patients who died did so within the first 48 hours from injury (80%). Regression analysis revealed that DELAY to IR was independently associated with more than a twofold higher risk of mortality (odds ratio 2.7, 95% confidence interval 1.6-4.9, p < 0.001). For every hour delay, the risk of mortality increased by 47%. These findings were independent of injury mechanism and most pertinent to level I trauma centers. CONCLUSION: In hemodynamically unstable trauma patients undergoing therapeutic catheter-based IR procedures, delay to IR was independently associated with more than a twofold higher risk of mortality. These results suggest that therapeutic IR procedures should be performed as expeditiously as possible and held to the same dogma as applied to definitive operative control of hemorrhage.

Surgeon volume and adequacy of thyroidectomy for differentiated thyroid cancer.

INTRODUCTION: We aimed to determine influence of surgeon volume on (1) frequency of appropriate initial surgery for differentiated thyroid cancer (DTC) and (2) completeness of resection. METHODS: We reviewed all initial thyroidectomies (Tx; lobectomy and total) performed in a health system during 2011; surgeons were grouped by number of Tx cases per year. For patients with histologic DTC ≥ 1 cm, surgeon volume was correlated with initial extent of the operation, and markers of complete resection including uptake on I(123) prescan, thyrotropin-stimulated thyroglobulin levels, and I(131) dose administered. RESULTS: Of 1,249 patients who underwent Tx by 42 surgeons, 29% had DTC ≥ 1 cm without distant metastasis. At a threshold of ≥ 30 Tx per year, surgeons were more likely to perform initial total Tx for DTC ≥ 1 cm (P = .01), and initial resection was more complete as measured by all 3 quantitative markers. For patients with advanced stage disease, a threshold of ≥ 50 Tx per year was needed before observing improvements in I(123) uptake (P = .004). CONCLUSION: Surgeons who perform ≥ 30 Tx a year are more likely to undertake the appropriate initial operation and have more complete initial resection for DTC patients. Surgeon volume is an essential consideration in optimizing outcomes for DTC patients, and even higher thresholds (≥ 50 Tx/year) may be necessary for patients with advanced disease.

Augmenting autophagy to treat acute kidney injury during endotoxemia in mice.

OBJECTIVE: To determine that 1) an age-dependent loss of inducible autophagy underlies the failure to recover from AKI in older, adult animals during endotoxemia, and 2) pharmacologic induction of autophagy, even after established endotoxemia, is of therapeutic utility in facilitating renal recovery in aged mice. DESIGN: Murine model of endotoxemia and cecal ligation and puncture (CLP) induced acute kidney injury (AKI). SETTING: Academic research laboratory. SUBJECTS: C57Bl/6 mice of 8 (young) and 45 (adult) weeks of age. INTERVENTION: Lipopolysaccharide (1.5 mg/kg), Temsirolimus (5 mg/kg), AICAR (100 mg/kg). MEASUREMENTS AND MAIN RESULTS: Herein we report that diminished autophagy underlies the failure to recover renal function in older adult mice utilizing a murine model of LPS-induced AKI. The administration of the mTOR inhibitor temsirolimus, even after established endotoxemia, induced autophagy and protected against the development of AKI. CONCLUSIONS: These novel results demonstrate a role for autophagy in the context of LPS-induced AKI and support further investigation into like interventions that have potential to alter the natural history of disease.

Necrotizing soft tissue infections.

BACKGROUND: Necrotizing soft tissue infections (NSTI) are an uncommon, but aggressive, problem with a potential for high morbidity and mortality rates. Establishing the diagnosis can be the major challenge. METHOD: Review of pertinent English-language literature. RESULTS: Early, aggressive surgical debridement combined with empiric broad-spectrum antibiotic therapy form the cornerstones of management. Novel therapeutic strategies such as hyperbaric oxygen, intravenous immunoglobulin, extracorporeal plasma treatment, and drotrecogin alfa (activated), also have been described, although their roles remain ill-defined. CONCLUSION: Early diagnosis, despite its difficulties, is essential to guide the implementation of appropriate life-saving therapies. Understanding the microbiology of NSTI is important, not only to guide antibiotic therapy, but also to provide insight into the pathogenesis of the disease that will permit the future development of rationally targeted interventions.

Molecular mediators of metastasis in head and neck squamous cell carcinoma.

BACKGROUND: The presence of regional metastasis in patients with head and neck squamous cell carcinoma (HNSCC) is a common and adverse event associated with poor prognosis and high mortality. Although significant improvements in standard therapies have increased the efficacy of local tumor management, the high incidence of tumor recurrence has resulted in limited improvements in overall survival rates. Understanding the molecular mechanisms that mediate HNSCC invasion and metastasis may enable identification of novel therapeutic targets for the prevention and management of tumor dissemination. METHODS: A literature review was performed. RESULTS: Several biologic mediators and mechanisms that have been implicated in HNSCC metastasis, such as cell adhesion molecules, proteolytic enzymes, growth factor signaling, metastasis suppressor genes, and chemokine receptors were reviewed. CONCLUSIONS: Prevention of HNSCC metastasis is an important clinical objective that requires an increased understanding of the molecular mechanisms of tumor invasion and dissemination.