Nicole E. Lopez

Targeting α-7 Nicotinic Acetylcholine Receptor in the Enteric Nervous System: A Cholinergic Agonist Prevents Gut Barrier Failure after Severe Burn Injury

We have previously shown that vagal nerve stimulation prevents intestinal barrier loss in a model of severe burn injury in which injury was associated with decreased expression and altered localization of intestinal tight junction proteins. α-7 Nicotinic acetylcholine receptor (α-7 nAchR) has been shown to be necessary for the vagus nerve to modulate the systemic inflammatory response, but the role of α-7 nAchR in mediating gut protection remained unknown. We hypothesized that α-7 nAchR would be present in the gastrointestinal tract and that treatment with a pharmacological agonist of α-7 nAchR would protect against burn-induced gut barrier injury. The effects of a pharmacological cholinergic agonist on gut barrier integrity were studied using an intraperitoneal injection of nicotine 30 minutes after injury. Intestinal barrier integrity was examined by measuring permeability to 4-kDa fluorescein isothiocyanate-dextran and by examining changes in expression and localization of the intestinal tight junction proteins occludin and ZO-1. Nicotine injection after injury prevented burn-induced intestinal permeability and limited histological gut injury. Treatment with nicotine prevented decreased expression and altered localization of occludin and ZO-1, as seen in animals undergoing burn alone. Defining the interactions among the vagus nerve, the enteric nervous system, and the intestinal epithelium may lead to development of targeted therapeutics aimed at reducing gut barrier failure and intestinal inflammation after severe injury.

Borderline resectable pancreatic cancer: Definitions and management

Pancreatic cancer is the fourth leading cause of cancer death in the United States. While surgical resection remains the only curative option, more than 80% of patients present with unresectable disease. Unfortunately, even among those who undergo resection, the reported median survival is 15-23 mo, with a 5-year survival of approximately 20%. Disappointingly, over the past several decades, despite improvements in diagnostic imaging, surgical technique and chemotherapeutic options, only modest improvements in survival have been realized. Nevertheless, it remains clear that surgical resection is a prerequisite for achieving long-term survival and cure. There is now emerging consensus that a subgroup of patients, previously considered poor candidates for resection because of the relationship of their primary tumor to surrounding vasculature, may benefit from resection, particularly when preceded by neoadjuvant therapy. This stage of disease, termed borderline resectable pancreatic cancer, has become of increasing interest and is now the focus of a multi-institutional clinical trial. Here we outline the history, progress, current treatment recommendations, and future directions for research in borderline resectable pancreatic cancer.

Improved Perioperative Outcomes With Minimally Invasive Distal Pancreatectomy: Results From a Population-Based Analysis

IMPORTANCE Interest in minimally invasive distal pancreatectomy (MIDP) has grown in recent years, but currently available data are limited. Greater insight into application patterns and outcomes may be gained from a national database inquiry. OBJECTIVES To study trends in the use of MIDP and compare the short-term outcomes of MIDP with those of open distal pancreatectomy. DESIGN, SETTING, AND PARTICIPANTS Population-based retrospective cohort study evaluating perioperative outcomes and hospital charge measures for distal pancreatectomy, comparing the surgical approaches and adjusting for patient- and hospital-level factors, among patients undergoing elective distal pancreatectomy from 1998 to 2009 in the Nationwide Inpatient Sample in a 20% stratified sample of all US hospitals. MAIN OUTCOMES AND MEASURES In-hospital mortality, rates of perioperative complications and splenectomy, total charges, and length of stay. RESULTS A total of 8957 distal pancreatectomies were included in this analysis, of which 382 (4.3%) were MIDPs. On a national level, this projected to 42,320 open distal pancreatectomies and 1908 MIDPs. The proportion of distal pancreatectomies performed via minimally invasive approaches tripled between 1998 and 2009, from 2.4% to 7.3%. The groups were comparable for sex and comorbidity profiles, while patients who underwent MIDP were 1.5 years older. On multivariate analysis, MIDP was associated with lower rates of overall predischarge complications, including lower incidences of postoperative infections and bleeding complications, as well as a shorter length of stay by 1.22 days. There were no differences in rates of in-hospital mortality, concomitant splenectomy, or total charges. CONCLUSIONS AND RELEVANCE This population-based study of MIDP reveals that the application of this approach has tripled in practice and provides strong evidence that MIDP has evolved into a safe option in the treatment of benign and malignant pancreatic diseases.

Vagal nerve stimulation protects cardiac injury by attenuating mitochondrial dysfunction in a murine burn injury model.

Mitochondria play a central role in the integration and execution of a wide variety of apoptotic signals. In the present study, we examined the deleterious effects of burn injury on heart tissue. We explored the effects of vagal nerve stimulation (VNS) on cardiac injury in a murine burn injury model, with a focus on the protective effect of VNS on mitochondrial dysfunction in heart tissue. Mice were subjected to a 30% total body surface area, full‐thickness steam burn followed by right cervical VNS for 10 min. and compared to burn alone. A separate group of mice were treated with the M3‐muscarinic acetylcholine receptor (M3‐AchR) antagonist 4‐DAMP or phosphatidylinositol 3 Kinase (PI3K) inhibitor LY294002 prior to burn and VNS. Heart tissue samples were collected at 6 and 24 hrs after injury to measure changes in apoptotic signalling pathways. Burn injury caused significant cardiac pathological changes, cardiomyocyte apoptosis, mitochondrial swelling and decrease in myocardial ATP content at 6 and 24 hrs after injury. These changes were significantly attenuated by VNS. VNS inhibited release of pro‐apoptotic protein cytochrome C and apoptosis‐inducing factor from mitochondria to cytosol by increasing the expression of Bcl‐2, and the phosphorylation level of Bad (pBad136) and Akt (pAkt308). These protective changes were blocked by 4‐DAMP or LY294002. We demonstrated that VNS protected against burn injury–induced cardiac injury by attenuating mitochondria dysfunction, likely through the M3‐AchR and the PI3K/Akt signalling pathways.

Ghrelin prevents disruption of the blood-brain barrier after traumatic brain injury.

Significant effort has been focused on reducing neuronal damage from post-traumatic brain injury (TBI) inflammation and blood-brain barrier (BBB)-mediated edema. The orexigenic hormone ghrelin decreases inflammation in sepsis models, and has recently been shown to be neuroprotective following subarachnoid hemorrhage. We hypothesized that ghrelin modulates cerebral vascular permeability and mediates BBB breakdown following TBI. Using a weight-drop model, TBI was created in three groups of mice: sham, TBI, and TBI/ghrelin. The BBB was investigated by examining its permeability to FITC-dextran and through quantification of perivascualar aquaporin-4 (AQP-4). Finally, we immunoblotted for serum S100B as a marker of brain injury. Compared to sham, TBI caused significant histologic neuronal degeneration, increases in vascular permeability, perivascular expression of AQP-4, and serum levels of S100B. Treatment with ghrelin mitigated these effects; after TBI, ghrelin-treated mice had vascular permeability and perivascular AQP-4 and S100B levels that were similar to sham. Our data suggest that ghrelin prevents BBB disruption after TBI. This is evident by a decrease in vascular permeability that is linked to a decrease in AQP-4. This decrease in vascular permeability may diminish post-TBI brain tissue damage was evident by decreased S100B.

A Comprehensive review of abdominal infections

Intra-abdominal infection (IAI) describes a diverse set of diseases. It is broadly defined as peritoneal inflammation in response to microorganisms, resulting in purulence in the peritoneal cavity[1]. IAI are classified as uncomplicated or complicated based on the extent of infection[2]. Uncomplicated abdominal infections involve intramural inflammation of the gastrointestinal (GI) tract without anatomic disruption. They are often simple to treat; however, when treatment is delayed or inappropriate, or the infection involves a more virulent nosocomial microbe, the risk of progression into a complicated abdominal infection becomes significant[3, 4]. Complicated abdominal infections extend beyond the source organ into the peritoneal space. They cause peritoneal inflammation, and are associated with localized or diffuse peritonitis[5]. Localized peritonitis often manifests as an abscess with tissue debris, bacteria, neutrophils, macrophages, and exudative fluid contained in a fibrous capsule. Diffuse peritonitis is categorized as primary, secondary or tertiary peritonitis. Primary peritonitis is also known as spontaneous bacterial peritonitis. It is thought to be the result of bacterial translocation across an intact gut wall[6]. These infections are commonly monomicrobial, and the infecting organism is primarily determined by patient demographics. For example, healthy young girls are most often infected by streptococcal organisms, cirrhotics by gram negative or enterococcal organisms, and peritoneal dialysis patients by Staphylococcus aureus[7, 8]. Diagnosis requires peritoneal fluid aspiration. Characteristics of infection include white blood cell count (WBC) > 500 cells/mm3, high lactate, and low glucose levels. Positive peritoneal fluid cultures are definitive, and resolution of infection is marked by peritoneal fluid with < 250 WBC/mm3[9]. Secondary peritonitis is caused by microbial contamination through a perforation, laceration, or necrotic segment of the GI tract[7]. Definitive diagnosis is based on clinical examination and history, and specific diagnoses can be confirmed by radiographic imaging[10]. If a patient is stable enough for transport, computed tomography (CT) scan with intravenous and oral contrast is the standard method of evaluating most intra-abdominal pathologies, such as appendicitis, diverticulitis, and colitis[11]. Suspected biliary pathology is the exception, and ultrasound is the preferred initial imaging modality for this spectrum of disease including acute cholecystitis, emphysematous cholecystitis, and cholangitis. Infections associated with secondary peritonitis are commonly polymicrobial and the infecting organisms are those most commonly associated with the source of contamination (see Table 1). Table 1 Expected organisms according to source Source Expected Organism Primary Peritonitis Young healthy female Streptococcus Cirrhotic Enteric gram negatives Enterococcus CAPD Staphylococcus aureus Secondary peritonitis Stomach and duodenum Streptococcus Lactobacillus Biliary E. coli, Klebsiella, Enterococcus Small Intestine E. coli, Klebsiella, Lactobacillus Streptococci Diptheroids Enterococci Distal ileum and colon Bacteroides fragilis Clostridium spp. E. coli Enterobacter spp. Klebsiella spp. Peptostreptococci Enterococci Teritiary peritonitis Enterococcus Candida Staphylococcus epidermidis Enterobacter Adapted from Weigelt JA [12]. Tertiary peritonitis represents an infection that is persistent or recurrent at least 48 hours after appropriate management of primary or secondary peritonitis. It is more common among critically ill or immunocompromised patients[12]. Because of the poor host defenses, it is also often associated with less virulent organisms, such as Enterococcus, Candida, Staphylococcus epidermidis, and Enterobacter[13]. Intra-abdominal sepsis is an IAI that results in severe sepsis or septic shock[2].

Early ghrelin treatment attenuates disruption of the blood brain barrier and apoptosis after traumatic brain injury through a UCP-2 mechanism.

Ghrelin has been shown to be anti-inflammatory and neuroprotective in models of neurologic injury. We hypothesize that treatment with ghrelin will attenuate breakdown of the blood brain barrier (BBB) and apoptosis 24h following traumatic brain injury (TBI). We believe this protection is at least in part mediated by up-regulation of UCP-2, thereby stabilizing mitochondria and preventing up-regulation of caspase-3. A weight drop model was used to create severe TBI. Balb/c mice were divided into 3 groups. Sham: no TBI or ghrelin treatment; TBI: TBI only; TBI/ghrelin: 20μg (IP) ghrelin at the time of TBI. BBB permeability to 70kDa FITC-Dextran was measured 24h following injury and quantified in arbitrary integrated fluorescence (afu). Brain tissue was subjected to TUNEL staining and TUNEL positive cells were quantified. Immunohistochemistry was performed on injured tissue to reveal patterns of caspase-3 and UCP-2 expression. TBI increased cerebral vascular permeability by three-fold compared to sham. Ghrelin treatment restored vascular permeability to the level of shams. TUNEL staining showed that ghrelin mitigated the significant increase in apoptosis that follows TBI. TBI increased both caspase-3 compared to sham. Treatment with ghrelin significantly increased UCP-2 compared to TBI alone and this increase in UCP-2 expression was associated with a decrease in expression of caspase-3. Early ghrelin treatment prevents TBI induced BBB disruption and TBI mediated apoptosis 24h following injury. These results demonstrate the neuroprotective potential of ghrelin as a therapy in TBI.

Cell surface localization and release of the candidate tumor suppressor Ecrg4 from polymorphonuclear cells and monocytes activate macrophages

We identified fresh human leukocytes as an abundant source of the candidate epithelial tumor suppressor gene, Ecrg4, an epigenetically regulated gene, which unlike other tumor suppressor genes, encodes an orphan‐secreted, ligand‐like protein. In human cell lines, Ecrg4 gene expression was low, Ecrg4 protein undetectable, and Ecrg4 promoter hypermethylation high (45–90%) and reversible by the methylation inhibitor 5‐AzaC. In contrast, Ecrg4 gene expression in fresh, normal human PBMCs and PMNs was 600–800 times higher than in cultured cell lines, methylation of the Ecrg4 promoter was low (<3%), and protein levels were readily detectable in lysates and on the cell surface. Flow cytometry, immunofluorescent staining, and cell surface biotinylation established that full‐length, 14‐kDa Ecrg4 was localized on PMN and monocyte cell surfaces, establishing that Ecrg4 is a membrane‐anchored protein. LPS treatment induced processing and release of Ecrg4, as detected by flow and immunoblotting, whereas an effect of fMLF treatment on Ecrg4 on the PMN cell surface was detected on the polarized R2 subpopulation of cells. This loss of cell surface Ecrg4 was associated with the detection of intact and processed Ecrg4 in the conditioned media of fresh leukocytes and was shown to be associated with the inflammatory response that follows severe, cutaneous burn injury. Furthermore, incubation of macrophages with a soluble Ecrg4‐derived peptide increased the P‐p65, suggesting that processing of an intact sentinel Ecrg4 on quiescent circulating leukocytes leads to processing from the cell surface following injury and macrophage activation.

Optimizing Surgical and Imatinib Therapy for the Treatment of Gastrointestinal Stromal Tumors

IntroductionThe discovery of activating KIT and PDGFRα mutations in gastrointestinal stromal tumors (GISTs) represented a milestone as it allowed clinicians to use tyrosine kinase inhibitors, like imatinib, to treat this sarcoma. Although surgery remains the only potentially curative treatment, patients who undergo complete resection may still experience local recurrence or distant metastases. Therapeutic strategies that combine surgical resection and adjuvant imatinib may represent the best treatment to maximize patient outcomes. In addition to the use of imatinib in the adjuvant and metastatic settings, neoadjuvant imatinib, employed as a cytoreductive therapy, can decrease tumor volume, increase the probability of complete resection, and may reduce surgery-related morbidities. Thus, selected patients with metastatic disease may be treated with a combination of preoperative imatinib and metastasectomy. However, it is critical that patients with GIST be evaluated by a multidisciplinary team to coordinate surgery and targeted therapy in order to maximize clinical outcomes.DiscussionFollowing a systematic literature review, we describe the presentation, diagnosis, and treatment of GIST, with a discussion of the risk assessment for imatinib therapy. The application of surgical options, combined with adjuvant/neoadjuvant or perioperative imatinib, and their potential impact on survival for patients with primary, recurrent, or metastatic GIST are discussed.

Vagal nerve stimulation decreases blood-brain barrier disruption after traumatic brain injury.

BACKGROUND Traumatic brain injury (TBI) may alter sympathetic tone causing autonomic abnormalities and organ dysfunction. Vagal nerve stimulation (VNS) has been shown to decrease inflammation and distant organ injury after TBI. It is unknown whether VNS may reduce blood-brain barrier (BBB) dysfunction after TBI. We hypothesize that VNS prevents TBI-induced breakdown of the BBB, subsequent brain edema, and neuronal injury. METHODS A weight-drop model was used to create severe TBI in balb/c mice. Animals were divided into three groups: TBI—TBI only; TBI or VNS—animals that were treated with 10 minutes of VNS immediately before TBI; and sham—animals with opening of the skull but no TBI and VNS treatment. Brain vascular permeability to injected (Mr 70,000) FITC-dextran was measured by radiated fluorescence 6 hours after injury. Injured tissue sections were stained for perivascular aquaporin 4 (AQP-4), an important protein causing BBB-mediated brain edema. Fluorescence was quantified under laser scanning by confocal microscopy. RESULTS Six hours after TBI, cerebral vascular permeability was increased fourfold compared with sham (mean [SD], 6.6E+08 [5.5E+07] arbitrary fluorescence units [afu] vs. 1.5E+08 [2.9E+07] afu; p < 0.001). VNS prevented the increase in permeability when compared with TBI alone (mean [SD], 3.5 E+08 [8.3E+07] afu vs. 6.6E+08 [5.5E+07] afu; p < 0.05). Perivascular expression of AQP-4 was increased twofold in TBI animals compared with sham (mean [SD], 0.96 [0.12] afu vs. 1.79 [0.37] afu; p < 0.05). Similarly, VNS decreased post-TBI expression of AQP-4 to levels similar to sham (mean [SD], 1.15 [0.12] afu; p < 0.05). CONCLUSION VNS attenuates cerebral vascular permeability and decreases the up-regulation of AQP-4 after TBI. Future studies are needed to assess the mechanisms by which VNS maintains the BBB.