Harmonization guidelines for HLA-peptide multimer assays derived from results of a large scale international proficiency panel of the Cancer Vaccine Consortium

Purpose: The Cancer Vaccine Consortium of the Cancer Research Institute (CVC-CRI) conducted a multicenter HLA-peptide multimer proficiency panel (MPP) with a group of 27 laboratories to assess the performance of the assay. Experimental design: Participants used commercially available HLA-peptide multimers and a well characterized common source of peripheral blood mononuclear cells (PBMC). The frequency of CD8+ T cells specific for two HLA-A2-restricted model antigens was measured by flow cytometry. The panel design allowed for participants to use their preferred staining reagents and locally established protocols for both cell labeling, data acquisition and analysis. Results: We observed significant differences in both the performance characteristics of the assay and the reported frequencies of specific T cells across laboratories. These results emphasize the need to identify the critical variables important for the observed variability to allow for harmonization of the technique across institutions. Conclusions: Three key recommendations emerged that would likely reduce assay variability and thus move toward harmonizing of this assay. (1) Use of more than two colors for the staining (2) collect at least 100,000 CD8 T cells, and (3) use of a background control sample to appropriately set the analytical gates. We also provide more insight into the limitations of the assay and identified additional protocol steps that potentially impact the quality of data generated and therefore should serve as primary targets for systematic analysis in future panels. Finally, we propose initial guidelines for harmonizing assay performance which include the introduction of standard operating protocols to allow for adequate training of technical staff and auditing of test analysis procedure

Prospective, population-based surveillance of the burden of Streptococcus pneumoniae in community-acquired pneumonia in older adults, Chrzanów County, Poland, 2010 to 2012

INTRODUCTION: Community-acquired pneumonia (CAP) caused by Streptococcus pneumoniae is a substantial cause of morbidity and mortality among older adults. This study estimated incidences of CAP, chest x-ray−confirmed CAP (CXR+CAP), S pneumonia- positive CAP, S pneumonia-positive CXR+CAP, and S. pneumoniae serotype distribution among 46,000 at-risk adults aged ≥ 50 years residing in Chrzanów County, Poland. MATERIAL AND METHODS: From January 2010 to January 2012, all facilities providing ambulatory and inpatient care enrolled all consenting resident patients with suspicion of CAP. Chest x-rays, urine, blood, and sputum samples were analyzed. Annualized incidence rates were determined. Presence of S pneumonia-positive CAP and/or S. pneumoniae serotype distribution was determined using the urine antigen detection assay (capable of detecting the serotypes in the 13-valent pneumococcal conjugate vaccine [PCV13]), BinaxNOW®, and/or microbiology cultures. RESULTS: Among 5055 enrolled patients, 1195 (23.7%) were diagnosed with CAP and 1166 (23.4%) had CXR+CAP. S. pneumoniae was detected in 144 (12.1%) and 131 (11.2%) patients from the CAP and CXR+CAP cohorts, respectively. Annualized incidence rates of CAP, CXR+CAP, S pneumonia-positive CAP, and S. pneumonia-positive CXR+CAP were 12.8, 12.5, 1.6, and 1.4 per 1000 residents, respectively. Among CXR+CAP patients, 39.7% were aged 50 to 64 years and 60.3% were aged ≥ 65 years. Incidence rates generally increased with age. The most common serotypes in S. pneumoniae-positive CXR+CAP patients were 3 (n = 15), 23F (n = 10), 18C (n = 9), and 9V (n = 6). CONCLUSIONS: CAP due to PCV13 serotypes is a source of morbidity among adults >50 years and may be reduced by greater access to pneumococcal vaccines.INTRODUCTION: Community-acquired pneumonia (CAP) caused by Streptococcus pneumoniae is a substantial cause of morbidity and mortality among older adults. This study estimated incidences of CAP, chest x-ray−confirmed CAP (CXR+CAP), S pneumonia- positive CAP, S pneumonia-positive CXR+CAP, and S. pneumoniae serotype distribution among 46,000 at-risk adults aged ≥ 50 years residing in Chrzanów County, Poland. MATERIAL AND METHODS: From January 2010 to January 2012, all facilities providing ambulatory and inpatient care enrolled all consenting resident patients with suspicion of CAP. Chest x-rays, urine, blood, and sputum samples were analyzed. Annualized incidence rates were determined. Presence of S pneumonia-positive CAP and/or S. pneumoniae serotype distribution was determined using the urine antigen detection assay (capable of detecting the serotypes in the 13-valent pneumococcal conjugate vaccine [PCV13]), BinaxNOW®, and/or microbiology cultures. RESULTS: Among 5055 enrolled patients, 1195 (23.7%) were diagnosed with CAP and 1166 (23.4%) had CXR+CAP. S. pneumoniae was detected in 144 (12.1%) and 131 (11.2%) patients from the CAP and CXR+CAP cohorts, respectively. Annualized incidence rates of CAP, CXR+CAP, S pneumonia-positive CAP, and S. pneumonia-positive CXR+CAP were 12.8, 12.5, 1.6, and 1.4 per 1000 residents, respectively. Among CXR+CAP patients, 39.7% were aged 50 to 64 years and 60.3% were aged ≥ 65 years. Incidence rates generally increased with age. The most common serotypes in S. pneumoniae-positive CXR+CAP patients were 3 (n = 15), 23F (n = 10), 18C (n = 9), and 9V (n = 6). CONCLUSIONS: CAP due to PCV13 serotypes is a source of morbidity among adults >50 years and may be reduced by greater access to pneumococcal vaccines

A critical assessment for the value of markers to gate-out undesired events in HLA-peptide multimer staining protocols

Background: The introduction of antibody markers to identify undesired cell populations in flow-cytometry based assays, so called DUMP channel markers, has become a practice in an increasing number of labs performing HLA-peptide multimer assays. However, the impact of the introduction of a DUMP channel in multimer assays has so far not been systematically investigated across a broad variety of protocols. Methods: The Cancer Research Institute%27s Cancer Immunotherapy Consortium (CRI-CIC) conducted a multimer proficiency panel with a specific focus on the impact of DUMP channel use. The panel design allowed individual laboratories to use their own protocol for thawing, staining, gating, and data analysis. Each experiment was performed twice and in parallel, with and without the application of a dump channel strategy. Results: The introduction of a DUMP channel is an effective measure to reduce the amount of non-specific MULTIMER binding to T cells. Beneficial effects for the use of a DUMP channel were observed across a wide range of individual laboratories and for all tested donor-antigen combinations. In 48% of experiments we observed a reduction of the background MULTIMER-binding. In this subgroup of experiments the median background reduction observed after introduction of a DUMP channel was 0.053%. Conclusions: We conclude that appropriate use of a DUMP channel can significantly reduce background staining across a large fraction of protocols and improve the ability to accurately detect and quantify the frequency of antigen-specific T cells by multimer reagents. Thus, use of a DUMP channel may become crucial for detecting low frequency antigen-specific immune responses. Further recommendations on assay performance and data presentation guidelines for publication of MULTIMER experimental data are provided

Cyclin D2-knock-out mice with attenuated dentate gyrus neurogenesis have robust deficits in long-term memory formation.

Neurobehavioral studies have produced contradictory findings concerning the function of neurogenesis in the adult dentate gyrus. Previous studies have proved inconsistent across several behavioral endpoints thought to be dependent on dentate neurogenesis, including memory acquisition, short-term and long-term retention of memory, pattern separation, and reversal learning. We hypothesized that the main function of dentate neurogenesis is long-term memory formation because we assumed that a newly formed and integrated neuron would have a long-term impact on the local neural network. We used a cyclin D2-knock-out (cyclin D2-/-) mouse model of endogenously deficient dentate neurogenesis to test this hypothesis. We found that cyclin D2-/- mice had robust and sustained loss of long-term memory in two separate behavioral tasks, Morris water maze (MWM) and touchscreen intermediate pattern separation. Moreover, after adjusting for differences in brain volumes determined by magnetic resonance (MR) imaging, reduced dentate neurogenesis moderately correlated with deficits in memory retention after 24 hours. Importantly, cyclin D2-/- mice did not show deficits in learning acquisition in a touchscreen paradigm of intermediate pattern separation or MWM platform location, indicating intact short-term memory. Further evaluation of cyclin D2-/- mice is necessary to confirm that deficits are specifically linked to dentate gyrus neurogenesis since cyclin D2-/- mice also have a reduced size of the olfactory bulb, hippocampus, cerebellum and cortex besides reduced dentate gyrus neurogenesis

Association Between Angiographic Complications and Clinical Outcomes Among Patients With Acute Coronary Syndrome Undergoing Percutaneous Coronary Intervention An EARLY ACS (Early Glycoprotein IIb/IIIa Inhibition in Non–ST-Segment Elevation Acute Coronary Syndrome) Angiographic Substudy

ObjectivesThe goal of this analysis was to determine the association between intraprocedural complications and clinical outcomes among patients with high-risk non–ST-segment elevation acute coronary syndrome (NSTEACS) undergoing percutaneous coronary intervention (PCI).BackgroundAmong patients undergoing PCI for NSTEACS, the relationship between intraprocedural complications and clinical outcomes, independent of epicardial and myocardial perfusion, has not been well characterized.MethodsThe EARLY ACS (Early Glycoprotein IIb/IIIa Inhibition in Non–ST-Segment Elevation Acute Coronary Syndrome) trial enrolled 9,406 patients with high-risk NSTEACS undergoing an early invasive strategy. Of these, 1,452 underwent angiographic assessment in an independent core laboratory and did not have a myocardial infarction (MI) between enrollment and angiography. We assessed the relationship between abrupt closure, loss of side branch(es), distal embolization, and no-reflow phenomenon and 30-day clinical outcomes in these patients.ResultsOf the patients, 166 (11.4%) experienced an intraprocedural complication. Baseline clinical characteristics were similar between patients who did and did not have complications. The 30-day composite of death or MI was significantly higher among patients with an intraprocedural complication (28.3% vs. 7.8%, odds ratio [OR]: 4.68, 95% confidence interval [CI]: 3.2 to 7.0, p < 0.001). Individually, both mortality (3.0% vs. 0.9%, OR: 3.60, 95% CI: 1.2 to 10.5, p = 0.019) and MI (27.1% vs. 7.4%, OR: 4.66, 95% CI: 3.1 to 7.0, p < 0.001) were significantly increased. After adjusting for differences in post-PCI epicardial and myocardial perfusion, the association with 30-day death or MI remained significant.ConclusionsAmong high-risk NSTEACS patients undergoing an invasive strategy, the incidence of intraprocedural complications is high, and the occurrence of these complications is associated with worse clinical outcomes independent of epicardial and myocardial perfusion.(Early Glycoprotein IIb/IIIa Inhibition in Patients With Non–ST-segment Elevation Acute Coronary Syndrome [EARLY ACS]; NCT00089895

Competition-induced stress does not explain deceptive alarm calling in tufted capuchin monkeys

Tactical deception has long attracted interest because it is often assumed to entail complex cognitive mechanisms. However, systematic evidence of tactical deception is rare and no study has attempted to determine whether such behaviours may be underpinned by relatively simple mechanisms. This study examined whether deceptive alarm calling among wild tufted capuchin monkeys, Cebus apella nigritus, feeding on contestable food resources can be potentially explained by a physiological mechanism, namely increased activation in the adrenocortex and the resulting production of glucocorticoids (GCs; ‘stress hormones’). This was tested experimentally in Iguazu? National Park, Argentina, by manipulating the potential for contest competition over food and noninvasively monitoring GC production through analysis of faecal hormone metabolites. If deceptive false alarms are indeed associated with adreno- cortical activity, it was predicted that the patterns of production of these calls would match the patterns of GC output, generally being higher in callers than noncallers in cases in which food is most contestable, and specifically being higher in callers on those occasions when a deceptive false alarm was produced. This hypothesis was not supported, as (1) GC output was significantly lower in association with the experimental introduction of contestable resources than in natural contexts wherein the potential for contest is lower, (2) within experimental contexts, there was a nonsignificant tendency for noncallers to show higher GC output than callers when food was most contestable, and (3) individuals did not show higher GC levels in cases in which they produced deceptive alarms relative to cases in which they did not. A learned association between the production of alarms and increased access to food may be the most likely cognitive explanation for this case of tactical deception, although unexplored physiological mechanisms also remain possible

Carbon Dynamics On The Louisiana Continental Shelf And Cross-Shelf Feeding Of Hypoxia

Large-scale hypoxia regularly develops during the summer on the Louisiana continental shelf. Traditionally, hypoxia has been linked to the vast winter and spring nutrient inputs from the Mississippi River and its distributary, the Atchafalaya River. However, recent studies indicate that much of the shelf ecosystem is heterotrophic. We used data from five late July shelfwide cruises from 2006 to 2010 to examine carbon and oxygen production and identify net autotrophic areas of phytoplankton growth on the Louisiana shelf. During these summer times of moderate river flows, shelfwide pH and particulate organic carbon (POC) consistently showed strong signals for net autotrophy in low salinity (\u3c25) waters near the river mouths. There was substantial POC removal via grazing and sedimentation in near-river regions, with 66–85 % of POC lost from surface waters in the low and mid-salinity ranges without producing strong respiration signals in surface waters. This POC removal in nearshore environments indicates highly efficient algal retention by the shelf ecosystem. Updated carbon export calculations for local estuaries and a preliminary shelfwide carbon budget agree with older concepts that offshore hypoxia is linked strongly to nutrient loading from the Mississippi River, but a new emphasis on cross-shelf dynamics emerged in this research. Cross-shelf transects indicated that river-influenced nearshore waters \u3c15 m deep are strong sources of net carbon production, with currents and wave-induced resuspension likely transporting this POC offshore to fuel hypoxia in adjacent mid-shelf bottom waters

Forming consensus to advance urobiome research

Urobiome research has the potential to advance the understanding of a wide range of diseases, including lower urinary tract symptoms and kidney disease. Many scientific areas have benefited from early research method consensus to facilitate the greater, common good. This consensus document, developed by a group of expert investigators currently engaged in urobiome research (UROBIOME 2020 conference participants), aims to promote standardization and advances in this field by the adoption of common core research practices. We propose a standardized nomenclature as well as considerations for specimen collection, preservation, storage, and processing. Best practices for urobiome study design include our proposal for standard metadata elements as part of core metadata collection. Although it is impractical to follow fixed analytical procedures when analyzing urobiome data, we propose guidelines to document and report data originating from urobiome studies. We offer this first consensus document with every expectation of subsequent revision as our field progresses

Rationale and Methods of the Study Protocol: Streptococcus pneumoniae Serotypes in Adults 18 Years and Older with Radiographically-Confirmed Community-Acquired Pneumonia (CAP)

This study was an active, prospective surveillance study of adults 18 years and older hospitalized with community-acquired pneumonia (CAP) due to Streptococcus pneumoniae conducted at 21 hospitals in ten cities across the United States. This report describes the surveillance methodology applied between October 7, 2013 and September 30, 2016, including the identification and description of surveillance areas and populations at-risk for CAP hospitalization for estimation of incidence rates for selected study sites