Flow Cytometry-based Cell Research Articles

Sensitive and broadly applicable residual disease detection in acute myeloid leukemia using flow cytometry-based leukemic cell enrichment followed by mutational profiling.

Persistent measurable residual disease (MRD) is an increasingly important prognostic marker in acute myeloid leukemia (AML). Currently, MRD is determined by multi‐parameter flow cytometry (MFC) or PCR‐based methods detecting leukemia‐specific fusion transcripts and mutations. However, while MFC is highly operator‐dependent and difficult to standardize, PCR‐based methods are only available for a minority of AML patients. Here we describe a novel, highly sensitive and broadly applicable method for MRD detection by combining MFC‐based leukemic cell enrichment using an optimized combinatorial antibody panel targeting CLL‐1, TIM‐3, CD123 and CD117, followed by mutational analysis of recurrently mutated genes in AML. In dilution experiments this method showed a sensitivity of 10−4 to 10−5 for residual disease detection. In prospectively collected remission samples this marker combination allowed for a median 67‐fold cell enrichment with sufficient DNA quality for mutational analysis using next generation sequencing (NGS) or digital PCR in 39 out of 41 patients. Twenty‐one samples (53.8%) tested MRD positive, whereas 18 (46.2%) were negative. With a median follow‐up of 559 days, 71.4% of MRD positive (15/21) and 27.8% (5/18) of MRD negative patients relapsed (P = .007). The cumulative incidence of relapse (CIR) was higher for MRD positive patients (5‐year CIR: 90.5% vs 28%, P < .001). In multivariate analysis, MRD positivity was a prominent factor for CIR. Thus, MFC‐based leukemic cell enrichment using antibodies against CLL‐1, TIM‐3, CD123 and CD117 followed by mutational analysis allows high sensitive MRD detection and is informative on relapse risk in the majority of AML patients.

Oxidative stress induced by piperine leads to apoptosis in Candida albicans.

Piperine inhibit Candida albicans growth (planktonic and biofilm) significantly in our study. Piperine exhibits excellent synergistic potential with fluconazole The proteome analysis suggests that piperine induced membrane damage leads to oxidative stress followed by cell cycle arrest and apoptosis.

Training Next-Generation Immunologists on GuavaÒ MuseÒ Cell Analyzer Platform

Abstract Immunology research in an academia setting is competitive and is especially challenging at any new professional program or new research laboratory. Faculty with start-up laboratories, welcome students into Biomedical Sciences research and in due course often seek for scientific collaborations. One such collaborative initiatives involved partnership between a non-profit academic research laboratory and Luminex, a biotechnology company that has a wide range of technological platforms including flow cytometers. This collaborative project is part of Research &amp; Scholarship course (also called R&amp;S course series) for PharmD students interested in Immunology research. The goal of the collaborative effort is to provide an opportunity for hands-on training of next generation immunologists on MuseÒ Cell Analyzer technology and proprietary kits/products designed for immunology and cell health analysis. R&amp;S students are trained by their research faculty on an innovative microcapillary-based benchtop flow cytometer. Enrollment into Immunology-based research projects would entail students to train with a faculty mentor, conduct literature review on the assigned project, critically think about a research hypothesis, design methods, perform data analysis, arrive at a reasonably valid conclusion, and present a poster showcasing their results at an R&amp;S poster day and/or other scientific conferences. Benchtop flow cytometry-based immunological and cell health assays relevant to R&amp;S students’ research project and their execution using kits from Luminex are presented.

Expansion of Neutrophils and Classical and Nonclassical Monocytes as a Hallmark in Relapsing-Remitting Multiple Sclerosis.

Neutrophils and monocytes encompassing the classical, intermediate, and nonclassical population constitute the majority of circulating myeloid cells in humans and represent the first line of innate immune defense. As such, changes in their relative and absolute amounts serve as sensitive markers of diverse inflammatory conditions. Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system, causing demyelination and axonal loss, affecting various neuron functions and often causing irreversible neurological disability. MS disease course is individually highly heterogeneous but can be classified as progressive (PMS) or relapsing-remitting (RRMS). Each MS course type may be further characterized as active or inactive, depending on the recent disability progression and/or current relapses. Data on specific alterations of the myeloid compartment in association with MS disease course are scarce and conflicting. In the current study, we systematically immunophenotyped blood myeloid leukocytes by flow cytometry in 15 healthy and 65 MS subjects. We found a highly significant expansion of granulocytes, CD15+ neutrophils, and classical and nonclassical monocytes in inactive RRMS (RRMSi) with concomitant shrinkage of the lymphocyte compartment, which did not correlate with biochemical readouts of systemic inflammation. Each of these leukocyte populations and the combined myeloid signature accurately differentiated RRMSi from other MS forms. Additionally, nonclassical monocyte proportions were particularly elevated in RRMSi individuals receiving disease-modifying therapy (DMT), such as natalizumab. Our results suggest that flow cytometry-based myeloid cell immunophenotyping in MS may help to identify RRMSi earlier and facilitate monitoring of DMT response.

Pingyangmycin inhibits glycosaminoglycan sulphation in both cancer cells and tumour tissues.

Pingyangmycin is a clinically used anticancer drug and induces lung fibrosis in certain cancer patients. We previously reported that the negatively charged cell surface glycosaminoglycans are involved in the cellular uptake of the positively charged pingyangmycin. However, it is unknown if pingyangmycin affects glycosaminoglycan structures. Seven cell lines and a Lewis lung carcinoma‐injected C57BL/6 mouse model were used to understand the cytotoxicity of pingyangmycin and its effect on glycosaminoglycan biosynthesis. Stable isotope labelling coupled with LC/MS method was used to quantify glycosaminoglycan disaccharide compositions from pingyangmycin‐treated and untreated cell and tumour samples. Pingyangmycin reduced both chondroitin sulphate and heparan sulphate sulphation in cancer cells and in tumours. The effect was persistent at different pingyangmycin concentrations and at different exposure times. Moreover, the cytotoxicity of pingyangmycin was decreased in the presence of soluble glycosaminoglycans, in the glycosaminoglycan‐deficient cell line CHO745, and in the presence of chlorate. A flow cytometry‐based cell surface FGF/FGFR/glycosaminoglycan binding assay also showed that pingyangmycin changed cell surface glycosaminoglycan structures. Changes in the structures of glycosaminoglycans may be related to fibrosis induced by pingyangmycin in certain cancer patients.

Refining cell-based assay to detect MOG-IgG in patients with central nervous system inflammatory diseases

BackgroundGiven that the spectrum of myelin oligodendrocyte glycoprotein immunoglobulin G (MOG-IgG) associated disease is yet to be fully defined, development of sensitive and highly specific assays to identify MOG-IgG is crucial to precisely define the clinical phenotypes, disease courses and prognosis to describe the full spectrum of MOG-IgG associated diseases. Here, we aim to validate a new in-house live cell-based assay (CBA) for screening MOG-IgG in patients with central nervous system inflammatory diseases. MethodsWe generated a full length MOG transfected HEK293 stable cell line using pIRES2-eGFP vector. Sera from 355 patients with central nervous system inflammatory diseases and 25 healthy individuals were evaluated for MOG-IgG seropositivity using in-house cell-based immunofluorescence assay (CBA-IF). The specificity of IgG (H + L) and IgG1-Fc secondary antibodies as well as IgM binding were determined by cell-based flow cytometry (CBA-FACS). The optimal cut-offs for determining seropositivity in CBA-FACS were calculated and the concordance of CBA-IF score and CBA-FACS was studied. The results of our CBA-IF were compared with the Oxford CBA-IF. Results11.5% (41/355) of patients were seropositive for MOG-IgG and had clinical phenotypes that were within the known clinical spectrum of MOG-IgG associated diseases. No typical multiple sclerosis patients, aquaporin-4-IgG positive neuromyelitis optica spectrum disorder or healthy individuals were MOG-IgG seropositive. Using CBA-FACS, the anti-human IgG (H + L) was found to be comparable to IgG1-Fc antibody. No IgM binding was observed in all the samples tested. CBA-IF score and CBA-FACS yielded high correlation. The concordance of the NCC CBA-IF with the Oxford CBA-IF was 98%. ConclusionWe have developed MOG-IgG CBAs that have different characteristics and benefits but with high specificity and concordance. The complementary use of two methods and follow-up study with larger cohort will increase the clinical usefulness of MOG-IgG CBAs.

The Aminoalkylindole BML-190 Negatively Regulates Chitosan Synthesis via the Cyclic AMP/Protein Kinase A1 Pathway in Cryptococcus neoformans.

Cryptococcus neoformans can cause fatal meningoencephalitis in patients with AIDS or other immunocompromising conditions. Current antifungals are suboptimal to treat this disease; therefore, novel targets and new therapies are needed. Previously, we have shown that chitosan is a critical component of the cryptococcal cell wall and is required for survival in the mammalian host and that chitosan deficiency results in rapid clearance from the mammalian host. We had also identified several specific proteins that were required for chitosan biosynthesis, and we hypothesize that screening for compounds that inhibit chitosan biosynthesis would identify additional genes/proteins that influence chitosan biosynthesis. To identify these compounds, we developed a robust and novel cell-based flow cytometry screening method to identify small-molecule inhibitors of chitosan production. We screened the ICCB Known Bioactives library and identified 8 compounds that reduced chitosan in C. neoformans We used flow cytometry-based counterscreens and confirmatory screens, followed by a biochemical secondary screen to refine our primary screening hits to 2 confirmed hits. One of the confirmed hits that reduced chitosan content was the aminoalkylindole BML-190, a known inverse agonist of mammalian cannabinoid receptors. We demonstrated that BML-190 likely targets the C. neoformans G-protein-coupled receptor Gpr4 and, via the cyclic AMP (cAMP)/protein kinase A (PKA) signaling pathway, contributes to an intracellular accumulation of cAMP that results in decreased chitosan. Our discovery suggests that this approach could be used to identify additional compounds and pathways that reduce chitosan biosynthesis and could lead to potential novel therapeutics against C. neoformansIMPORTANCECryptococcus neoformans is a fungal pathogen that kills ∼200,000 people every year. The cell wall is an essential organelle that protects fungi from the environment. Chitosan, the deacetylated form of chitin, has been shown to be an essential component of the cryptococcal cell wall during infection of a mammalian host. In this study, we screened a set of 480 compounds, which are known to have defined biological activities, for activity that reduced chitosan production in C. neoformans Two of these compounds were confirmed using an alternative method of measuring chitosan, and one of these was demonstrated to impact the cAMP signal transduction pathway. This work demonstrates that the cAMP pathway regulates chitosan biosynthesis in C. neoformans and validates that this screening approach could be used to find potential antifungal agents.

A method for high-purity isolation of neutrophil granulocytes for functional cell migration assays

Abstract Background Neutrophil-mediated killing of pathogens is one of the most significant functions of the primary defense of the host. Neutrophil activity and migration play a key role in inflammatory conditions. To gain insights into the interactions between neutrophils and neutrophil migration-related disorders, a large number of sophisticated methods have been developed. The technical limitations of isolating highly purified neutrophil populations, minimizing both cell death and activation during the isolation process, and the short lifespan of neutrophils present challenges for studying specific functions of neutrophils in vitro. In this study, we aimed to evaluate a separation medium-based density gradient method to obtain highly purified neutrophil populations and combined this protocol with a model for studying neutrophil migration in-vitro. Materials and methods Human granulocytes were isolated using Lympholyte-poly solution. The purity and viability of isolated neutrophils were assessed by flow cytometry and morphological analysis. Neutrophil activation was confirmed by immunocytochemistry. Lastly, filter assay was performed to measure neutrophil chemotaxis. Results and discussion All validation experiments revealed that this method was capable of generating a highly purified neutrophil population for further functional in-vitro assays. Consequently, this study demonstrates a quick, cost effective, and easy-to-follow model, and may be a significant alternative to isolation methods that need extra subsequent steps such as flow cytometry-based cell sorting for reaching highly purified neutrophil population. Conclusion The suggested combination of methods for the isolation and cell migration analysis of human neutrophils is highly recommended to use for disease models involving neutrophil migration such as autoinflammatory disorders.

Abstract NT-092: FUNCTIONAL HETEROGENEITY OF ACQUIRED PARP INHIBITOR RESISTANCE IN BRCA1-DEFICIENT CELLS

Abstract INTRODUCTION: More than 50% of high-grade serous ovarian cancers (HGSOC) are deficient in Homologous Recombination (HR) DNA repair, predominantly due to inactivation of BRCA1 or BRCA2 genes. HR-deficient cancers are sensitive to inhibitors of Poly-ADP Ribose Polymerase (PARP), and PARP inhibitors (PARPi's) have shown promising efficacy in the treatment of HGSOC. However, the majority of HGSOC patients will eventually develop resistance to PARPi's, and no overall survival benefit has been reported. Clinically, best characterized mechanism of PARPi resistance is restoration of BRCA1/2 protein expression accounting for 20-50% of clinical cases. Recently, emerging preclinical evidence has emphasized the role of replication fork protection in PARPi resistance. In addition, transcriptional regulation of the DNA damage response (DDR) proteins, including 53BP1, have been shown to contribute to PARPi resistance. However, the lack of understanding on the functional heterogeneity of the different PARP inhibitor resistance mechanisms has hampered the clinical targeting and development of biomarkers for acquired PARPi resistance in HGSOC. RESULTS: To understand the dynamics and origin of PARPi resistance, we generated a TP53-/- and BRCA1-/- deficient epithelial (RPE) cell line using the CRISPR/Cas9 system. These cells are deficient in HR, and therefore are hypersensitive to PARPi in vitro. Using cyclic exposure of increasing concentrations of the PARPi Niraparib, we selected a cell population that were resistant to high concentrations (µM range) of the drug. We next isolated single cell clones from the resistant pool and performed deep functional profiling. As expected, the clones were resistant to multiple PARPi, including Niraparib, Olaparib and Talazoparib, and showed decreased DNA damage after PARPi treatment. However, none of the clones has restored BRCA1 protein expression. In functional assays, some clones had different levels of restored HR, and others showed a predominantly fork-stable, non HR-restored phenotype. Upon cytogenetic analyses, the parental RPETP53-/-BRCA1-/- cells exhibited high clonal heterogeneity in terms of ploidy, whereas the PARPi resistant clones are mostly triploid. Interestingly, the HR-restored clones have lower levels of baseline chromosomal aberrations; however, mitomycin C induced chromosomal breaks in all the clones. In flow cytometry-based cell cycle profiling, the clones retained the G2/M accumulation upon PARPi treatment, similar to the parental RPETP53-/-BRCA1-/- cells. Interestingly, all PARPi resistant clones show decreased levels of the DDR protein KAP1, and divergent expression levels of other DDR proteins, such as 53BP1. Importantly, the clones show significant heterogeneity in terms of sensitivity to cisplatin, as well as to DDR targeting agents, such as inhibitors of ATR and CHK1. CONCLUSIONS: We have engineered BRCA1- deficient cells that are resistant to PARPi, and show that subclones of from these cells have significant functional heterogeneity in DNA repair dynamics, and are differentially vulnerable to DDR targeting agents. Importantly, our model system suggests that acquired PARP inhibitor resistance involves the adoption of several distinct resistance mechanisms that are potentially linked to DDR regulation. To enable clinical translation of our findings, we are in the process of performing genomic and transcriptomic profiling to discover the genomic evolution, common vulnerabilities, and novel biomarkers for acquired PARPi resistance in HGSOC. Citation Format: Anniina Färkkilä, Alfredo Rodriquez, Jaana Olkkonen, Larissa Sambel, Julieta Dominiquez, Niraj Joshi, Connor Clairmont, Jia Zhou, Kah-Suan Lim, Sampsa Hautaniemi, and Alan D'Andrea. FUNCTIONAL HETEROGENEITY OF ACQUIRED PARP INHIBITOR RESISTANCE IN BRCA1-DEFICIENT CELLS [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr NT-092.

In Vivo Assessment of Intracellular Dynamics Comparing Injection Versus Oral Azacitidine in a Phase IIb Investigator Initiated Clinical Trial

In Vivo Assessment of Intracellular Dynamics Comparing Injection Versus Oral Azacitidine in a Phase IIb Investigator Initiated Clinical Trial

1179A novel exonic variant in TRIM55 gene predisposes to heart failure

Abstract Background/Introduction Heart failure is a potentially lethal cardiac disorder. The genetic variants as well as the molecular signaling pathways accounting for the majority of the heritability of sporadic heart failure are unknown. Here, we screened for novel genetic variants associated with heart failure in the Finnish population. A previously uncharacterized variant in TRIM55 was selected for further validation. Purpose To identify and characterize heart failure-associated genetic variants. Methods Heart failure-associated genetic variants were screened from the FINRISK survey study data by the National Institute of Health and Welfare of Finland (genotype data available for approx. 28,000 individuals, 1,485 individuals with heart failure). A probably damaging heart failure-associated exonic variant in TRIM55 gene (rs138811034), encoding an E140K variant, was functionally characterized in HL-1 mouse cardiomyocytes using CRISPR/Cas9 technology to introduce the alteration in the endogenous locus. Variant cell lines were analyzed with RNA sequencing. The effect of the variant on cellular viability was tested with CTG assays. Hypertrophic response after isoproterenol treatment was studied by quantitating cell surface area with immunofluorescence microscopy. Western analyses of p21 expression and flow cytometry-based cell cycle analyses were carried out to assess effects on cell cycle progression. In zebrafish embryos in vivo, cardiac contractility was videomicroscopically measured after transient CRISPR-mediated knockdown of the trim55a gene. Results HL-1 cells harboring Trim55 E140K/− genotype demonstrated significantly reduced viability as compared to the WT/WT cell (P=0.006), similarly to the cell line with double knockout (−/−) of the gene. E140K/−, −/− and WT/− cell lines demonstrated a significant hypertrophic response to isoproterenol stimulation (P=0.007, P=0.007 and P=0.018, respectively), while the WT/WT cells did not. In the E140K/− and −/− cell lines, p21 protein expression was increased and there were significantly fewer cells in the mitotic G2/M phase of the cell cycle, as compared to the WT/WT cells (P=0.031 and P=0.024, respectively). In the E140K/− and −/− cell lines, RNA sequencing revealed systemic alterations in the genes related to cardiac contractility and stress. In zebrafish embryos with deleted trim55a gene, ejection fraction was reduced as compared to control sgRNA-injected embryos (30.8% ± 6.0% vs 43.9%± 2.2% (95% CIs), respectively, P=0.019). Conclusions TRIM55 E140K variant was statistically associated with heart failure in the Finnish population. In HL-1 cardiomyocytes in vitro, the variant reduced cellular viability and cell cycle progression and promoted hypertrophy. In zebrafish embryos, loss of trim55a caused a significant reduction in cardiac contractility. These findings are consistent with a novel role for TRIM55 polymorphism in predisposing to heart failure. Acknowledgement/Funding Finnish Cultural Foundation (Varsinais-Suomi Regional Fund), Inkeri ja Mauri Vänskän säätiö, Academy of Finland [310507], Sigrid Jusélius Foundation

Targeted in-vitro-stimulation reveals highly proliferative multi-virus-specific human central memory T cells as candidates for prophylactic T cell therapy.

Adoptive T cell therapy (ACT) has become a treatment option for viral reactivations in patients undergoing allogeneic hematopoietic stem cell transplantation (alloHSCT). Animal models have shown that pathogen-specific central memory T cells (TCM) are protective even at low numbers and show long-term survival, extensive proliferation and high plasticity after adoptive transfer. Concomitantly, our own recent clinical data demonstrate that minimal doses of purified (not in-vitro- expanded) human CMV epitope-specific T cells can be sufficient to clear viremia. However, it remains to be determined if human virus-specific TCM show the same promising features for ACT as their murine counterparts. Using a peptide specific proliferation assay (PSPA) we studied the human Adenovirus- (AdV), Cytomegalovirus- (CMV) and Epstein-Barr virus- (EBV) specific TCM repertoires and determined their functional and proliferative capacities in vitro. TCM products were generated from buffy coats, as well as from non-mobilized and mobilized apheresis products either by flow cytometry-based cell sorting or magnetic cell enrichment using reversible Fab-Streptamers. Adjusted to virus serology and human leukocyte antigen (HLA)-typing, donor samples were analyzed with MHC multimer- and intracellular cytokine staining (ICS) before and after PSPA. TCM cultures showed strong proliferation of a plethora of functional virus-specific T cells. Using PSPA, we could unveil tiniest virus epitope-specific TCM populations, which had remained undetectable in conventional ex-vivo-staining. Furthermore, we could confirm these characteristics for mobilized apheresis- and GMP-grade Fab-Streptamer-purified TCM products. Consequently, we conclude that TCM bare high potential for prophylactic low-dose ACT. In addition, use of Fab-Streptamer-purified TCM allows circumventing regulatory restrictions typically found in conventional ACT product generation. These GMP-compatible TCM can now be used as a broad-spectrum antiviral T cell prophylaxis in alloHSCT patients and PSPA is going to be an indispensable tool for advanced TCM characterization during concomitant immune monitoring.

Remission clone in acute myeloid leukemia shows growth advantage after chemotherapy but is distinct from leukemic clone

In a previously published case study of acute myeloid leukemia, we tracked the dynamics of somatic mutations over 9 years. Interestingly, we observed a group of mutations that expanded during remission, which we named the "remission clone." To determine the nature of the remission clones, we performed flow cytometry-based cell sorting followed by ultradeep sequencing. The remission clone repeatedly expanded after chemotherapeutic cycles and was suppressed during relapse in the myeloid lineage (multipotent hematopoietic stem, progenitor, and myeloid cells). On the other hand, the remission clone was consistently observed in lymphoid lineages (B and T cells) regardless of the disease state. When transfected into the HEK-293 cell line, the NR2C2(A93V) mutant exhibited a growth advantage (all p values < 0.05). The results indicate that the remission clone seems to be another form of clonal hematopoiesis, but without a clear association with leukemia. As the remission clone is present in both myeloid and lymphoid lineages, it likely originates from ancestral hematopoietic cell lineages. More importantly, the remission clone is distinct from the leukemic clone; therefore, mutations expanded during remission require special interpretation when performing next-generation sequencing-based measurable residual disease assessment.

Cardamonin, a natural chalcone, reduces 5-fluorouracil resistance of gastric cancer cells through targeting Wnt/β-catenin signal pathway.

Objectives Cardamonin (CD), an active chalconoid, has been extensively studied in a wide variety of human tumors. However, the effects and underlying mechanism of cardamonin on 5-fluorouracil (5-FU)-resistant gastric cancer (GC) remain largely unclear. This study aimed to investigate the antitumor effects of cardamonin on 5-FU-resistant GC cells and explore the molecular mechanisms underlying its therapeutic potential. Methods The antitumor activities of cardamonin, 5-FU and their combination against BGC-823 and BGC-823/5-FU cells were determined using cytotoxicity assay, flow cytometry-based cell cycle analysis and Annexin V apoptosis assay. The effect of cardamonin on P-glycoprotein activity was assessed by Rh123 uptake assay. Real-time PCR, Western blotting and Co-immunoprecipitation analysis were carried out to assess the inhibition of Wnt/β-catenin signaling pathway. A xenograft mouse model was established using BALB/c nude mice to examine the combinatorial effects of cardamonin and 5-FU on tumor growth. Results Our data provided the first demonstration that cardamonin significantly enhanced the chemosensitivity of 5-FU in GC cells via suppression of Wnt/β-catenin signaling pathway. Additionally, the combination of cardamonin and 5-FU might result in the apoptosis and cell cycle arrest of BGC-823/5-FU cells, accompanied by the downregulated expression levels of P-glycoprotein, β-catenin and TCF4. More importantly, our results demonstrated that cardamonin specifically disrupted the formation of β-catenin/TCF4 complex, leading to TCF4-mediated transcriptional activation in 5-FU-resistant GC cells. Besides, through a xenograft mouse model, co-administration of cardamonin and 5-FU significantly retarded tumor growth in vivo, thus, confirming our in vitro findings. Conclusions Overall, this study revealed that cotreatment of cardamonin and 5-FU could strongly potentiate the antitumor activity of 5-FU, and put forth cardamonin as a rational therapeutic strategy for drug-resistant GC treatment.

Histone acetyltransferase CBP is critical for conventional effector and memory T-cell differentiation in mice

Compared with naïve T cells, memory CD8+ T cells have a transcriptional landscape and proteome that are optimized to generate a more rapid and robust response to secondary infection. Additionally, rewired kinase signal transduction pathways likely contribute to the superior recall response of memory CD8+ T cells, but this idea has not been experimentally confirmed. Herein, we utilized an MS approach to identify proteins that are phosphorylated on tyrosine residues in response to Listeria-induced T-cell receptor (TCR) stimulation in both naïve and memory CD8+ T cells from mice and separated by fluorescence- and flow cytometry-based cell sorting. This analysis identified substantial differences in tyrosine kinase signaling networks between naïve and memory CD8+ T cells. We also observed that an important axis in memory CD8+ T cells couples Janus kinase 2 (JAK2) hyperactivation to the phosphorylation of CREB-binding protein (CBP). Functionally, JAK2-catalyzed phosphorylation enabled CBP to bind with higher affinity to acetylated histone peptides, indicating a potential epigenetic mechanism that could contribute to rapid initiation of transcriptional programs in memory CD8+ T cells. Moreover, we found that CBP itself is essential for conventional effector and memory CD8+ T-cell formation. These results indicate how signaling pathways are altered to promote CD8+ memory cell formation and rapid responses to and protection from repeat infections.

Flow Cytometry-Based Cell Type-Specific Assessment of Target Regulation by Pulmonary siRNA Delivery.

Pulmonary siRNA delivery has attracted strong interest and has been reported to successfully mediate target gene knockdown in a number of disease models. However, the nature of the epithelial cells that eventually take up siRNA and the question if other lung cell types may also be transfected has so far been neglected. Therefore, we describe here a flow cytometry-based method using transgenic enhanced green fluorescence protein (EGFP) expressing mice for the differentiation of transfected lung cell populations based on their antigen expression.

622. Increased IgA Coating of Gut Microbes After Administration of Killed, Whole-Cell Oral Cholera Vaccine

BackgroundCholera vaccines are recommended for use in outbreaks to prevent infections and reduce severity of disease. Variable immune responses are observed after administration of killed, whole-cell cholera vaccines, and limited data suggest that the gut microbiome may be one factor influencing immune responses to vaccination. Methods. We used microbial DNA sequencing of stool and serum vibriocidal titers to examine the gut microbiome and immune responses to vaccination at day 0, 7, 17 and 44 in adult vaccine recipients in Dhaka, Bangladesh. Using flow cytometry-based bacterial cell sorting, we identified IgA-coated gut microbes in stool before and after vaccination in a subset of patients. Results. Vibriocidal titer magnitude and kinetics were used to classify participants. Within 17 days of vaccination, 86/89 (96%) adults developed a four-fold rise in vibriocidal titer. Gut microbial diversity was not significantly changed after vaccination. Rate of seroconversion (four-fold increase in vibriocidal titer by Day 3 after vaccination) was faster in participants with increased bacteria from the genus Prevotella (multivariate analysis using linear models, q value 0.04). The gut microbes of participants with higher peak vibriocidal titers was characterized by increased Prevotella (3% vs. <0.1% of the total microbiome, P < 0.001 unpaired t-test, linear discriminant analysis score >3.5), particularly the species Prevotella copri (P < 0.001, unpaired t-test, linear discriminant analysis score >3.5). Lipopolysaccharide from Prevotella species is known to increase vaccination-associated antigen-specific antibody titers in animal models. Additionally, IgA coating of gut microbes in stool increased after vaccination, from 8.9% IgA coated at baseline to a peak level of 19% during follow-up (Wilcoxon signed rank test, P < 0.01). Conclusion. Certain microbiome profiles are correlated with greater immune responses to cholera vaccination, and IgA coating of gut bacteria indicates which commensal species may be participating in the mucosal immune response. The potential for modulation of mucosal immune responses based on gut microbial species warrants further study.Disclosures All authors: No reported disclosures.

Silica/Polyethylene Glycol Hybrid Materials Prepared by a Sol-Gel Method and Containing Chlorogenic Acid.

Chlorogenic acid (CGA) is a very common dietary polyphenolic compound. CGA is becoming very attractive due to its potential use as preventive and therapeutic agent in many diseases, including cancer. Inorganic/organic hybrid materials are gaining considerable attention in the biomedical field. The sol-gel process provides a useful way to obtain functional organic/inorganic hybrids. The aim of this study was to synthesize silica/polyethylene glycol (PEG) hybrids with different percentages of CGA by sol-gel technique and to investigate their impact on the cancer cell proliferation. Synthesized materials have been chemically characterized through the FTIR spectroscopy and their bioactivity evaluated looking by SEM at their ability to produce a hydroxyapatite layer on their surface upon incubation with simulated body fluid (SBF). Finally, their effects on cell proliferation were studied in cell lines by direct cell number counting, MTT, flow cytometry-based cell-cycle and cell death assays, and immunoblotting experiments. Notably, we found that SiO2/PEG/CGA hybrids exhibit clear antiproliferative effects in different tumor, including breast cancer and osteosarcoma, cell lines in a CGA dependent manner, but not in normal cells. Overall, our results increase the evidence of CGA as a possible anticancer agent and illustrate the potential for clinical applications of sol-gel synthesized SiO2/PEG/CGA materials.

Detection of Antibodies That Neutralize the Cellular Uptake of Enzyme Replacement Therapies with a Cell-based Assay.

The administration of enzyme replacement therapies (ERTs) and other biologic therapies to patients may elicit an anti-drug immune response. The characterization of these anti-drug antibodies (ADA), especially those that may neutralize the biological activity of the drug, termed neutralizing antibodies (NAbs), is crucial in understanding the effects of these antibodies on the drug's pharmacological profile. This protocol describes a cell-based flow cytometry method to detect factors that neutralize the cellular uptake of a representative lysosomal ERT in human matrix. The protocol consists of three procedures: screening, a confirmatory step, and titer assays to detect, identify, and establish the relative level of neutralizing antibody titer in subject samples.In this method, samples are first mixed with the fluorophore-conjugated ERT product, then incubated with cells [e.g., human T lymphocytes (Jurkat cells)] that express a cell-surface cation-independent mannose 6-phosphate receptor (CI-M6PR), and finally, analyzed with a flow cytometer. A sample without NAbs will result in the uptake of the fluorophore-conjugated ERT product via CI-M6PR, whereas, the presence of NAbs will bind to the drug and interfere with the CI-M6PR binding and uptake. The amount of the fluorophore-conjugated ERT internalized by the Jurkat cells is measured by flow cytometry and evaluated as the percentage (%) signal inhibition compared to the response obtained in the presence of a representative drug-naïve matrix. In the confirmatory step, the samples are pre-incubated with ERT-conjugated magnetic beads to deplete drug-specific factors that bind to the drug (such as NAbs) prior to an incubation with cells. Samples that screen and confirm positive for drug-specific NAbs in the assay are then serially diluted to generate an antibody titer. Semi-quantitative antibody titers may be correlated with measurements of drug safety and efficacy.

Detection of Antibodies That Neutralize the Cellular Uptake of Enzyme Replacement Therapies with a Cell-based Assay

The administration of enzyme replacement therapies (ERTs) and other biologic therapies to patients may elicit an anti-drug immune response. The characterization of these anti-drug antibodies (ADA), especially those that may neutralize the biological activity of the drug, termed neutralizing antibodies (NAbs), is crucial in understanding the effects of these antibodies on the drug's pharmacological profile. This protocol describes a cell-based flow cytometry method to detect factors that neutralize the cellular uptake of a representative lysosomal ERT in human matrix. The protocol consists of three procedures: screening, a confirmatory step, and titer assays to detect, identify, and establish the relative level of neutralizing antibody titer in subject samples. In this method, samples are first mixed with the fluorophore-conjugated ERT product, then incubated with cells [e.g., human T lymphocytes (Jurkat cells)] that express a cell-surface cation-independent mannose 6-phosphate receptor (CI-M6PR), and finally, analyzed with a flow cytometer. A sample without NAbs will result in the uptake of the fluorophore-conjugated ERT product via CI-M6PR, whereas, the presence of NAbs will bind to the drug and interfere with the CI-M6PR binding and uptake. The amount of the fluorophore-conjugated ERT internalized by the Jurkat cells is measured by flow cytometry and evaluated as the percentage (%) signal inhibition compared to the response obtained in the presence of a representative drug-naïve matrix. In the confirmatory step, the samples are pre-incubated with ERT-conjugated magnetic beads to deplete drug-specific factors that bind to the drug (such as NAbs) prior to an incubation with cells. Samples that screen and confirm positive for drug-specific NAbs in the assay are then serially diluted to generate an antibody titer. Semi-quantitative antibody titers may be correlated with measurements of drug safety and efficacy.