Analysis of Immune Landscape in Pancreatic and Ileal Neuroendocrine Tumours Demonstrates an Immune Cold Tumour Microenvironment.

INTRODUCTION: Neuroendocrine tumours (NETs) are rare tumours with an increasing incidence. While low- and intermediate-grade pancreatic NET (PanNET) and small intestinal NET (siNET) are slow growing, they have a relatively high rate of metastasizing to the liver, leading to substantially worse outcomes. In many solid tumours, the outcome is determined by the quality of the antitumour immune response. However, the quality and significance of antitumour responses in NETs are incompletely understood. This study provides clinico-pathological analyses of the tumour immune microenvironment in PanNET and siNETs. METHODS: Formalin-fixed paraffin-embedded tissue from consecutive resected PanNETs (61) and siNETs (131) was used to construct tissue microarrays (TMAs); 1-mm cores were taken from the tumour centre, stroma, tumour edge, and adjacent healthy tissue. TMAs were stained with antibodies against CD8, CD4, CD68, FoxP3, CD20, and NCR1. T-cell counts were compared with counts from lung cancers. RESULTS: For PanNET, median counts were CD8+ 35.4 cells/mm2, CD4+ 7.6 cells/mm2, and CD68+ macrophages 117.7 cells/mm2. For siNET, there were CD8+ 39.2 cells/mm2, CD4+ 24.1 cells/mm2, and CD68+ 139.2 cells/mm2. The CD8+ cell density in the tumour and liver metastases were significantly lower than in the adjacent normal tissues, without evidence of a cell-rich area at the tumour edge that might have suggested immune exclusion. T-cell counts in lung cancer were significantly higher than those in PanNET and siNETs: CD8+ 541 cells/mm2 and CD4+ 861 cells/mm2 (p ≤ 0.0001). CONCLUSION: PanNETs and siNETs are immune cold with no evidence of T cell exclusion; the low density of immune infiltrates indicates poor antitumour immune responses.

Predicting Ventriculoperitoneal Shunt Dependence in High Grade Aneurysmal Subarachnoid Hemorrhage.

INTRODUCTION: Aneurysmal subarachnoid hemorrhage (aSAH) commonly presents with hydrocephalus due to obstruction of cerebrospinal fluid (CSF) passage across the ventricular system in the brain. Placement of an external ventricular device (EVD) and in some cases ventriculoperitoneal shunt (VPS) are often necessary for patients requiring prolonged CSF diversion. The study aimed at evaluating critical factors that play a role in determining the need for extended extraventricular drainage. METHODS: We performed a retrospective observational cohort study of two groups of patients with radiological imaging confirmed high grade aSAH (Hunt & Hess grades 3-5) who required VPS placement, shunt-dependent group, and who did not require long term CSF diversion, non-shunt-dependent group. We collected and analyzed data regarding the daily CSF output for 10 days following EVD placement, daily EVD height, intracranial pressure (ICP) and cerebral perfusion pressure (CPP), indicators of hydrocephalus, and CSF characteristics. RESULTS: The cohort, comprising of 8 patients in the shunt-dependent group and 32 patients in the non-shunt-dependent group, displayed median daily CSF output of 275.1 mL/day and 193.4 mL/day, respectively (P = .0005). ROC curve for CSF drainage for the two groups showed an area under the curve (AUC) of 0.71 with a 95% confidence interval (CI) 0.65 to 0.77. Qualitative analysis of CSF characteristics revealed that the shunt-dependent group had more proteinaceous, darker red color, and greater proportion of red blood cells (RBCs) although not statistically significant. CONCLUSIONS: Determinants of prolonged CSF drainage requirements in patients with high grade aSAH are not fully elucidated to this date and there is no standardized protocol for CSF diversion. Our study revealed potential markers that can be used in the assessment for the need for long term CSF diversion. Our limited sample size necessitates further research to establish clear correlations and cutoffs of these parameters in predicting long term CSF diversion requirements.

Utility of KI-67 as a prognostic biomarker in pulmonary neuroendocrine neoplasms: a systematic review and meta-analysis.

OBJECTIVES: Ki-67, a marker of cellular proliferation, is associated with prognosis across a wide range of tumours, including gastroenteropancreatic neuroendocrine neoplasms (NENs), lymphoma, urothelial tumours and breast carcinomas. Its omission from the classification system of pulmonary NENs is controversial. This systematic review sought to assess whether Ki-67 is a prognostic biomarker in lung NENs and, if feasible, proceed to a meta-analysis. RESEARCH DESIGN AND METHODS: Medline (Ovid), Embase, Scopus and the Cochrane library were searched for studies published prior to 28 February 2019 and investigating the role of Ki-67 in lung NENs. Eligible studies were those that included more than 20 patients and provided details of survival outcomes, namely, HRs with CIs according to Ki-67 percentage. Studies not available as a full text or without an English manuscript were excluded. This study was prospectively registered with PROSPERO. RESULTS: Of 11 814 records identified, seven studies met the inclusion criteria. These retrospective studies provided data for 1268 patients (693 TC, 281 AC, 94 large cell neuroendocrine carcinomas and 190 small cell lung carcinomas) and a meta-analysis was carried out to estimate a pooled effect. Random effects analyses demonstrated an association between a high Ki-67 index and poorer overall survival (HR of 2.02, 95% CI 1.16 to 3.52) and recurrence-free survival (HR 1.42; 95% CI 1.01 to 2.00). CONCLUSION: This meta-analysis provides evidence that high Ki-67 labelling indices are associated with poor clinical outcomes for patients diagnosed with pulmonary NENs. This study is subject to inherent limitations, but it does provide valuable insights regarding the use of the biomarker Ki-67, in a rare tumour. PROSPERO REGISTRATION NUMBER: CRD42018093389.

Optimization of Detergent-Mediated Reconstitution of Influenza A M2 Protein into Proteoliposomes.

We report the optimization of detergent-mediated reconstitution of an integral membrane-bound protein, full-length influenza M2 protein, by direct insertion into detergent-saturated liposomes. Detergent-mediated reconstitution is an important method for preparing proteoliposomes for studying membrane proteins, and must be optimized for each combination of protein and membrane constituents used. The purpose of the reconstitution was to prepare samples for site-directed spin-labeling electron paramagnetic resonance (SDSL-EPR) studies. Our goals in optimizing the protocol were to minimize the amount of detergent used, reduce overall proteoliposome preparation time, and confirm the removal of all detergent. The liposomes were comprised of (1-palmitoyl-2-oleyl-sn-glycero-phosphocholine (POPC) and 1-palmitoyl-2-oleyl-sn-glycero-3-[phospho-rac-(1-glycerol)] (POPG), and the detergent octylglucoside (OG) was used for reconstitution. Rigorous physical characterization was applied to optimize each step of the reconstitution process. We used dynamic light scattering (DLS) to determine the amount of OG needed to saturate the preformed liposomes. During detergent removal by absorption with Bio-Beads, we quantified the detergent concentration by means of a colorimetric assay, thereby determining the number of Bio-Bead additions needed to remove all detergent from the final proteoliposomes. We found that the overnight Bio-Bead incubation used in previously published protocols can be omitted, reducing the time needed for reconstitution. We also monitored the size distribution of the proteoliposomes with DLS, confirming that the size distribution remains essentially constant throughout the reconstitution process.

Concurrent visualization in a production supercomputing environment.

We describe a concurrent visualization pipeline designed for operation in a production supercomputing environment. The facility was initially developed on the NASA Ames "Columbia" supercomputer for a massively parallel forecast model (GEOS4). During the 2005 Atlantic hurricane season, GEOS4 was run 4 times a day under tight time constraints so that its output could be included in an ensemble prediction that was made available to forecasters at the National Hurricane Center. Given this time-critical context, we designed a configurable concurrent pipeline to visualize multiple global fields without significantly affecting the runtime model performance or reliability. We use MPEG compression of the accruing images to facilitate live low-bandwidth distribution of multiple visualization streams to remote sites. We also describe the use of our concurrent visualization framework with a global ocean circulation model, which provides a 864-fold increase in the temporal resolution of practically achievable animations. In both the atmospheric and oceanic circulation models, the application scientists gained new insights into their model dynamics, due to the high temporal resolution animations attainable.

Marked improvement in nocturnal gastroesophageal reflux in a large cohort of patients with obstructive sleep apnea treated with continuous positive airway pressure.

BACKGROUND: Nocturnal gastroesophageal reflux (nGER) is common in patients with obstructive sleep apnea (OSA). Small, short-term studies have shown that treatment with nasal continuous positive airway pressure (CPAP) decreases esophageal acid exposure. OBJECTIVE: To examine the relationship between OSA and nGER, and the effect of CPAP on nGER, in a long-term follow-up study of a large cohort of patients with OSA and nGER. METHODS: We prospectively studied 331 patients diagnosed as having OSA between October 1, 1993, and November 30, 2000. At baseline, patients graded their frequency of nGER symptoms on a scale of 1 (never) to 5 (always). All patients were prescribed CPAP for their OSA. At follow-up, the frequency of nGER symptoms was obtained by telephone interview. RESULTS: Of the 331 patients with OSA, nGER was present in 204 (62%) before treatment with CPAP. Follow-up was obtained in 181 patients (89%). Of these 181 patients, 165 (91%) were still using CPAP and 16 (9%) were not, forming the treatment and control groups, respectively. The patients compliant with CPAP had a significant improvement in nGER score, from a mean of 3.38 before CPAP treatment to 1.75 after treatment (48% improvement; P<.001), while patients not using CPAP (control subjects) showed no improvement (mean, 3.56 to 3.44; P =.55). There was a strong correlation between CPAP pressure and improvement in nGER score (correlation, r = 0.70; P<.001), with patients with higher CPAP pressures demonstrating a greater improvement in nGER score. CONCLUSIONS: Nocturnal GER is common in patients with OSA. Treatment with nasal CPAP decreases the frequency of nGER symptoms by 48%. Higher nasal CPAP pressures are associated with greater improvement in nGER.

C-terminal juxtamembrane region of full-length M2 protein forms a membrane surface associated amphipathic helix.

The influenza A M2 protein is a 97-residue integral membrane protein involved in viral budding and proton conductance. Although crystal and NMR structures exist of truncated constructs of the protein, there is disagreement between models and only limited structural data are available for the full-length protein. Here, the structure of the C-terminal juxtamembrane region (sites 50-60) is investigated in the full-length M2 protein using site-directed spin-labeling electron paramagnetic resonance (EPR) spectroscopy in lipid bilayers. Sites 50-60 were chosen for study because this region has been shown to be critical to the role the M2 protein plays in viral budding. Continuous wave EPR spectra and power saturation data in the presence of paramagnetic membrane soluble oxygen are consistent with a membrane surface associated amphipathic helix. Comparison between data from the C-terminal juxtamembrane region in full-length M2 protein with data from a truncated M2 construct demonstrates that the line shapes and oxygen accessibilities are remarkably similar between the full-length and truncated form of the protein.

Augmented transforming growth factor-beta1 in gingival crevicular fluid of smokers with chronic periodontitis.

BACKGROUND: Patients who smoke are at increased risk for chronic periodontitis (CP). Also, CP patients who smoke exhibit significantly less reduction of probing depths and gains in clinical attachment compared to non-smokers following periodontal therapy. Several studies suggest that the effects of smoking on the host response may be paramount in regulating the basal systemic inflammatory status and therapeutic outcomes in this cohort. Growth factors, specifically transforming growth factor beta1 (TGF-beta1), are critical in regulating the wound healing response by controlling cell division, differentiation, and motility. The hypothesis to be tested was that gingival crevicular fluid (GCF) TGF-beta1 production was altered in smokers compared to non-smokers with CP. METHODS: GCF was collected from smokers and non-smokers with CP, both at baseline and 1 to 2 weeks after initial therapy. GCF volume was determined using an electronic device and TGF-beta1 concentration was measured by enzyme-linked immunosorbent assay (ELISA). RESULTS: Smokers exhibited a higher mean concentration of GCF TGF-beta1 at baseline compared to non-smokers (P = 0.03). After initial therapy, smokers exhibited significantly less reduction in mean GCF volume compared to non-smokers (P = 0.04). CONCLUSIONS: Augmented constitutive production of GCF TGF-beta1 in smokers may explain the clinical appearance of fibrotic gingival tissue exhibited by this patient cohort. A diminished reduction in GCF volume in smokers following root instrumentation suggests a chronic inflammatory status in conjunction with an ineffective host response. These findings support the concept that smokers with CP display an altered local inflammatory response after initial therapy, perhaps symptomatic of colonization by residual periodontal pathogens.

A pathologist's survey on the reporting of sessile serrated adenomas/polyps.

AIM: The purpose of this survey was to ascertain reporting habits of pathologists towards sessile serrated adenomas/polyps (SSA/P). METHODS: A questionnaire designed to highlight diagnostic criteria, approach and clinical implications of SSA/P was circulated electronically to 45 pathologists in the UK and North America. RESULTS: Forty-three of 45 pathologists agreed to participate. The vast majority (88%) had a special interest in gastrointestinal (GI) pathology, had great exposure to GI polyps in general with 40% diagnosing SSA/P at least once a week if not more, abnormal architecture was thought by all participants to be histologically diagnostic, and 11% would make the diagnosis if a single diagnostic histological feature was present in one crypt only, while a further 19% would diagnose SSA/P in one crypt if more than one diagnostic feature was present. The vast majority agreed that deeper sections were useful and 88% did not feel proliferation markers were useful. More than one-third did not know whether, or did not feel that, their clinicians were aware of the implications of SSA/P. CONCLUSIONS: 98% of pathologists surveyed are aware that SSA/P is a precursor lesion to colorectal cancer, the majority agree on diagnostic criteria, and a significant number feel that there needs to be greater communication and awareness among pathologists and gastroenterologists about SSA/P.

Accuracy of visual assessments of proliferation indices in gastroenteropancreatic neuroendocrine tumours.

AIMS: To compare the accuracy of eyeball estimates of the Ki-67 proliferation index (PI) with formal counting of 2000 cells as recommend by the Royal College of Pathologists. METHODS: Sections from gastroenteropancreatic neuroendocrine tumours were immunostained for Ki-67. PI was calculated using three methods: (1) a manual tally count of 2000 cells from the area of highest nuclear labelling using a microscope eyepiece graticule; (2) eyeball estimates made by four pathologists within the same area of highest nuclear labelling; and (3) image analysis of microscope photographs taken from this area using the ImageJ 'cell counter' tool. ImageJ analysis was considered the gold standard for comparison. RESULTS: Levels of agreement between methods were evaluated using Bland-Altman plots. Agreement between the manual tally and ImageJ assessments was very high at low PIs. Agreement between eyeball assessments and ImageJ analysis varied between pathologists. Where data for low PIs alone were analysed, there was a moderate level of agreement between pathologists' estimates and the gold standard, but when all data were included, agreement was poor. CONCLUSIONS: Manual tally counts of 2000 cells exhibited similar levels of accuracy to the gold standard, especially at low PIs. Eyeball estimates were significantly less accurate than the gold standard. This suggests that tumour grades may be misclassified by eyeballing and that formal tally counting of positive cells produces more reliable results. Further studies are needed to identify accurate clinically appropriate ways of calculating.

Lower gastrointestinal bleeding--management.

Although acute LGIB is only about one fifth as common and is usually less hemodynamically significant than upper gastrointestinal bleeding, it presents numerous unique clinical challenges. The best diagnostic approach for patients with active bleeding is unknown, but urgent prepared colonoscopy is safe and likely to be beneficial (Fig. 3, Table 2). In patients who have aggressive bleeding or recurrent bleeding, it is critical for the practitioner to judge when angiography and surgery are necessary.

Ischemic colitis: a clinical review.

Ischemic colitis is the most common form of intestinal ischemia. It manifests as a spectrum of injury from transient self-limited ischemia involving the mucosa and submucosa to acute fulminant ischemia with transmural infarction that may progress to necrosis and death. Although there are a variety of causes, the most common mechanism is an acute, self-limited compromise in intestinal blood flow. Patients typically have mild abdominal pain and tenderness over the involved segment of bowel. There is usually passage of blood mixed with stool, but hemodynamically significant bleeding is unusual. Although computed tomography may have suggestive findings, colonoscopy is the procedure of choice for diagnosis. Supportive care with intravenous fluids, optimization of hemodynamic status, avoidance of vasoconstrictive drugs, bowel rest, and empiric antibiotics will produce clinical improvement within 1 to 2 days in most patients. Twenty percent of patients will have development of peritonitis or may deteriorate despite conservative management and will require surgery.

Urgent colonoscopy for evaluation and management of acute lower gastrointestinal hemorrhage: a randomized controlled trial.

OBJECTIVES: We hypothesized that early intervention in patients with lower gastrointestinal bleeding (LGIB) would improve outcomes and therefore conducted a prospective randomized study comparing urgent colonoscopy to standard care. METHODS: Consecutive patients presenting with LGIB without upper or anorectal bleeding sources were randomized to urgent purge preparation followed immediately by colonoscopy or a standard care algorithm based on angiographic intervention and expectant colonoscopy. RESULTS: A total of 50 patients were randomized to each group. A definite source of bleeding was found more often in urgent colonoscopy patients (diverticula, 13; angioectasia, 4; colitis, 4) than in the standard care group (diverticula, 8; colitis, 3) (the odds ratio for the difference among the groups was 2.6; 95% CI 1.1-6.2). In the urgent colonoscopy group, 17 patients received endoscopic therapy; in the standard care group, 10 patients had angiographic hemostasis. There was no difference in outcomes among the two groups-including: mortality 2%versus 4%, hospital stay 5.8 versus 6.6 days, ICU stay 1.8 versus 2.4 days, transfusion requirements 4.2 versus 5 units, early rebleeding 22%versus 30%, surgery 14%versus 12%, or late rebleeding 16%versus 14% (mean follow-up of 62 and 58 months). CONCLUSION: Although urgent colonoscopy identified a definite source of LGIB more often than a standard care algorithm based on angiography and expectant colonoscopy, the approaches are not significantly different with regard to important outcomes. Thus, decisions concerning care for patients with acute LGIB should be based on individual experience and local expertise.

The perception of cancer risk in patients with prevalent Barrett's esophagus enrolled in an endoscopic surveillance program.

BACKGROUND & AIMS: Patients with Barrett's esophagus (BE) have a risk of esophageal adenocarcinoma of approximately 0.5% per year. Patients may have difficulty understanding this risk. This study assessed the perceived risk of cancer in patients with BE, and correlated their risk estimates with their health care use behaviors. METHODS: We performed a survey of patients with BE participating in an endoscopic surveillance program at 2 sites: a university teaching hospital and a Veterans' Administration hospital. A questionnaire also elicited their demographics as well as their sources of health information. Health care behaviors, including physician visits and endoscopic surveillance behaviors, were assessed. Patients were classified as either overestimators or nonoverestimators of risk. Characteristics of overestimators, as well as health care use patterns, were assessed. RESULTS: One hundred eighteen patients met entry criteria, and 92 (78%) completed all the questionnaires. Sixty-eight percent of patients overestimated their 1-year risk of cancer, with a mean estimated 1-year cancer risk being 13.6%. The lifetime risk also was overestimated by 38% of patients. Patients who overestimated risk were more likely to be Veterans' Administration medical center patients, have more symptomatic reflux, and were more likely to use the Internet to get health care information. There was no significant difference in physician visits between overestimators and nonestimators (1.2 visits per year vs 1.0, P = .20), nor in endoscopy use (5.7 endoscopies per 5-year period vs 5.0, P = .42). CONCLUSIONS: The majority of patients with prevalent BE participating in an endoscopic surveillance program overestimated their chances of developing adenocarcinoma of the esophagus. Efforts to improve education of such patients with BE are warranted.

Gastrointestinal endoscopic evaluation of premenopausal women with iron deficiency anemia.

GOALS: To evaluate whether the gastrointestinal tract could be a source of chronic blood loss in premenopausal women with iron deficiency anemia. BACKGROUND: While premenopausal women with iron deficiency anemia are typically managed with simple iron replacement, the standard of care for postmenopausal women and men is to exclude a gastrointestinal source of bleeding. STUDY: We identified 111 premenopausal women who underwent endoscopy for the sole indication of iron deficiency anemia. RESULTS: The mean age was 42.5 years. Lesions potentially causative of iron deficiency anemia were detected in 22 patients (20%). Upper gastrointestinal lesions were present in 14 patients (13%) and included only erosive lesions. Lower gastrointestinal lesions were detected in 8 patients (7.2%) and included colon cancer (2.7%), inflammatory bowel disease (3.6%), and a colonic ulcer >1 cm (0.9%). Patients with upper gastrointestinal lesions were more likely to use aspirin or nonsteroidal antiinflammatory drugs (11/14, 79%) than patients with no lesions (26/89, 23%; P = 0.043). Occult blood was more common in patients with lower gastrointestinal lesions 8/8 (100%) and patients with upper gastrointestinal lesions (9/14, 64%) than in patients without lesions (28/89, 31%; P = 0.037 and 0.039). Gastrointestinal symptoms were significantly more common in patients with gastrointestinal lesions than in patients without lesions. CONCLUSIONS: A gastrointestinal source of chronic blood loss was identified in a substantial proportion of premenopausal women with iron deficiency anemia. Patients with gastrointestinal symptoms, fecal occult blood, and/or weight loss should undergo endoscopy.

Acute gastrointestinal bleeding.

Acute gastrointestinal bleeding is a common disorder with a wide spectrum of presentations that may encompass multiple clinical scenarios. Initial hemodynamic assessment and resuscitation are critical. Once accomplished, the source of bleeding should be localized, active bleeding should be stopped, the underlying abnormality should be treated, and recurrent bleeding should be prevented. The means to accomplish these goals depends on the specific clinical situation. For most forms of upper gastrointestinal bleeding, early endoscopy is the cornerstone of diagnosis and management. It can predict and improve clinical outcomes. A variety of endoscopic and pharmacologic modalities are effective at achieving and maintaining hemostasis. The optimum means of evaluation and treatment of acute lower gastrointestinal bleeding is less clear and is now evolving. Endoscopy (usually expectant, less often early) is widely used and effective for diagnosis but has unproven therapeutic benefits. Angiography is effective (diagnostically and/or therapeutically) in certain situations. Surgery offers the opportunity for definitive therapy at the cost of higher morbidity. At this time, the approach to evaluation and management should be based on the specific clinical situation and available local expertise.