Congratulations to the top 4 honorees who will present their work at the Summit 2014!:
1. Immunophenotyping of neutrophil subpopulations during Salmonella Typhimurium Colitis. Stefan Jellbauer #,1,2, Janet Liu#,1,2, Suzanne Klaus1,2, Robert A. Edwards3, Thomas A. Lane4, and Manuela Raffatellu1,2
#contributed equally,1 Department of Microbiology and Molecular Genetics,2 Institute for Immunology, 3Department of Pathology and Laboratory Medicine, 4Department of Molecular Biology and Biochemistry. University of California, Irvine.
The presence of functional immune cells in the intestinal mucosa determines whether a pathogen like Salmonella Typhimurium causes inflammatory diarrhea or potentially fatal bacteremia. Clinical and experimental evidence strongly suggests the characteristic influx of neutrophils into the intestinal mucosa during S. Typhimurium infection is important in preventing its dissemination. Neutrophils constitute one of the first lines of the innate immune response. However, there are no detailed studies about the characteristics of neutrophils that are recruited to control S. Typhimurium. Here we present a flow cytometry and imaging flow cytometry based approach to immunophenotype these distinct subpopulations of neutrophils during S. Typhimurium colitis.
Consistent with a protective role of neutrophils during infection, increased dissemination of S. Typhimurium to the mesenteric lymph nodes and spleen was observed in neutrophil-depleted animals. Neutrophil antimicrobial protein levels in cecal tissue were decreased in anti-CXCR2 treated animals, but anti-Gr-1 treated animals showed similar levels of cecal antimicrobial proteins as mock-depleted, infected animals. Remarkably, histopathological analysis showed the opposite trend regarding the presence of neutrophils in the cecum: no difference between anti-CXCR2-treated mice versus mock-treated, infected mice, but fewer neutrophils in the ceca of anti-Gr-1 treated animals. Flow cytometric analysis confirmed the relative depletion of infiltrating neutrophils, while showing different protein expression patterns in intestinal neutrophils than bone marrow or blood neutrophils. In particular, we found that neutrophils migrating to the gut upon S. Typhimurium infection may be either positive or negative for the cytosolic antimicrobial protein calprotectin, whose expression appears to be inducible in neutrophils. Specific neutrophil subtypes can also produce Th17 cytokines.
These results suggest that there may be distinct, previously unrecognized populations of neutrophils in the gut mucosa during S. Typhimurium infection, some of which migrating to the gut in a CXCR2-dependent fashion.
2. RECEPTOR OCCUPANCY AND INTERNALIZATION OF AN ANTI-IL-7 RECEPTOR ANTIBODY. Brent Kern(1), Eugenia Kraynov (1), Li-Fen Lee (2), and Chad Ray (1)
(1) Pharmacokinetics, Dynamics, and Metabolism, Pfizer, La Jolla, California, USA, (2) Experimental Medicine, Pfizer, San Francisco, California, USA
In vitro (or ex vivo) quantification of cellular parameters is often employed to inform systems models that define the relationship between drug exposure and response. We developed two flow cytometry based assays to evaluate the interaction between the IL-7 receptor (IL-7R) and an anti-IL-7R monoclonal antibody (Ab1). IL-7R occupancy and rate of internalization in the presence of Ab1 was determined using flow cytometry in monkeys and human whole blood. Two commercially available antibodies were used to detect IL-7R. One detected “free” and the other determined “total” receptor. Receptor internalization was measured ex vivo at various time points at 37oC. The receptor occupancy assay was used in vivo following administration of Ab1 into cynomolgus monkeys at 0.3, 3, and 30 mg/kg. The amount of Ab1 bound to receptor and the total amount of receptor was quantified. The ex vivo receptor occupancy mean IC50 values in monkey and human whole blood were 5.64 ± 2.38 and 9.39 ± 1.54 ng/ml, respectively. The ex vivo rate of internalization of Ab1-IL-7R complex was characterized by a slope of 384 ± 24 and 2482 ± 319, for monkey and human, respectively. In the in vivo monkey study, no free IL-7R was detectable on the CD3 T cell surface at 0.25 hr post-dose in all treatment groups. Free receptor levels returned to 78% on day 8 at 0.3 mg/kg, 55% on day 15 at 3 mg/kg, and 12% on day 15 at 30 mg/kg, compared to pre-dose levels. By Day 22, free IL-7R levels in all dose groups returned to their baseline. Ab1 treatment resulted in a significant reduction in total IL-7R. Effective biomeasures provide a link between species and the ex vivo and in vivo systems. Integration of these values into systems models reduces the number of model assumptions and increases the likelihood of success.
3.Digitally-synthesized beat frequency multiplexing for ultra-high throughput imaging flow cytometry. Eric D. Diebold, Brandon W. Buckley, and Bahram Jalali.
Departments of Electrical Engineering & Biomedical Engineering, UCLA
Fluorescence imaging flow cytometry provides undoubted advantages compared to traditional flow cytometry based on single-point measurements. The ability to localize the distribution of fluorescent probes within cells flowing at high speeds enables detailed high-content cell-level analysis of large cell populations, as well as high sensitivity and specificity rare cell detection. However, the weak optical emission of fluorescent probes and the trade-off between imaging speed and sensitivity is highly problematic for acquiring blur-free images of cells moving at the meter per second velocities common in most flow cytometers. Here, we present a technique to overcome these limitations that employs approaches common in the field of wireless communications in order to generate blur-free fluorescence images of cells in flow. Deemed fluorescence imaging using radiofrequency-tagged emission (FIRE), this approach to fluorescence microscopy maps the image into the radiofrequency spectrum using the beating of digitally-synthesized optical fields. A single photomultiplier tube simultaneously detects the image fluorescence from hundreds of spatial points in parallel, and the signal is de-multiplexed using massively parallel digital lock-in amplification to form an image. FIRE combines the benefits of photomultiplier tube sensitivity and speed with acousto-optic-based frequency-domain multiplexing, radiofrequency spectrum digital synthesis, and digital lock-in amplification to enable diffraction-limited fluorescence imaging of cells at a throughput of approximately 50,000 cells per second. This work represents more than an order of magnitude improvement in speed over the current state-of-the-art in fluorescence imaging flow cytometry.
4. Co-lyophilization of PGD2 assay control and quantum dot nanocrystals to trace experimental variables and analytes in a Whole Blood Flow Cytometric Assay. Christine M. Evangelista1, John Thomas1, John Dunne2†, John Ferbas1 and Shelley Sims Belouski1
1 Department of Medical Sciences, Amgen, Thousand Oaks, CA, 2 Formerly of BD Biosciences, San Jose, CA, †Current affiliation Owl Biomedical, Santa Barbara, CA
Custom assays can provide meaningful biomarker data to clinical studies but can be limited by logistical challenges that introduce variability. We have previously reported a flow cytometric (FCM) assay that stimulates fresh whole blood specimens with prostaglandin D2 (PGD2) to induce down regulation of the CRTH2 receptor. The inhibitory effect of investigational CRTH2 antagonist therapies can be assessed within the method by comparing unstimulated to a predefined low and high concentration of PGD2 stimulated whole blood specimens drawn at key pharmacokinetic time points throughout the study.
To decrease the variability of this method performed at the clinical sites, we implemented lyophilization and nanocrystal technologies. Plates were pre-loaded with lyophilized excipient only, low and high PGD2 to control concentration and preserve biologic activity. Unique quantum dot Qdot® nanocrystals were included in each of the lyophilized preparations to code the PGD2 dose identity of the wells. Results demonstrate our ability to trace or decode each PGD2 treated or untreated sample in whole blood throughout the analysis chain. Moreover, the addition of Qdot® nanocrystals to the wells had no impact on assay performance. Therefore, reagent co-lyophilization with pre-defined Qdot® nanocrystal combinations presents a favorable approach to maximize reagent stability and eliminate sample misidentification in clinical FCM assays.
Poster Session competition! Have your research efforts recognized!
SoCal Flow invites all undergraduate and graduate students, post-docs and non-management rank professionals (industry or academic) to submit an abstract to the Poster Session at SUMMIT 2014, March 24-25 at the Beckman Center, UC Irvine, CA.
Abstracts should be no longer than 300 words and are due March 3rd, 2014.
Abstracts must include title, names of contributing authors and author affiliations. The name of the presenting author must be lead/first author. Submit your abstract to lbrown at coh.org
Applicants must register for the meeting at socalflow.org . Registration opens January 2014.
Up to four posters will be selected for excellence in cytometry. The honorees will be invited to present their work in an Oral Symposium at the SUMMIT and will receive a registration fee refund and a $100 honorarium. The abstract will also be highlighted in the conference program.
Honorees will be announced March 7th, 2014.
Posters size is limited to 48in x 48in. Poster session flier.