Background: Blood pressure (BP) variability is an independent risk factor for cardiovascular disease (CVD). BP variability has been associated with cardiovascular organ damage and is a potential therapeutic target for the prevention of CVD. Recently, the gut microbiome (GM) has been shown to have a pathological role in hypertension, however, the association between GM and BP variability is poorly understood. Methods: 241 community-dwelling individuals free of symptomatic CVD from Hong Kong (113 males and 128 females, mean age 54±6 years) underwent ambulatory BP monitoring and stool microbiota shotgun sequencing. BP variability was determined as systolic/diastolic BP (SBP/DBP) coefficient of variation (CoV), nighttime dipping, and morning BP surge (MBPS). Sleep latency was estimated using 7-day Actigraphy data. Associations of BP variability with GM, and plasma and stool short-chain fatty acids (SCFAs) were analyzed under statistical models adjusting for age, sex, serum glucose and lipids, sodium intake assessed by urine analysis, menopause status, smoking, fatty liver, BP and sleep latency. Results: Women had a significantly higher 24-hour SBP ( p =0.0005) and DBP ( p =0.002) CoV than men. 24-hour SBP CoV had a negative association with GM α-diversity (Shannon and Simpson’s index: all P <0.05) and a positive association with Firmicutes/Bacteroidetes ratio ( P <0.05), suggesting gut dysbiosis in participants with higher systolic BP variability. Several GM species and SCFAs were significantly associated with the indices of BP variability in both men and women. Notably, Parabacteroides merdae had a negative association with both systolic and diastolic BP CoV (all P <0.05-0.01). Bacteroidetes dorei and Bacteroides intestinalis were reduced in women with higher SBP CoV ( P <0.05) and nondipping status ( P< 0.01), respectively. Prewaking and sleepthrough MBPS had a positive association with Faecalibacterium prausnitzii in women (all P <0.001) and plasma acetic acid levels in men ( P <0.05-0.001). Further analysis indicated that Bacteroidetes dorei may mediate SBP CoV via plasma iso-butyric acid in women (bootstrapping 95% CI: -3.6 to -0.19; P <0.05). Conclusions: This cross-sectional study suggests sex-specific associations between GM and BP variability. Most noteworthy, higher systolic BP variability was associated with a significant reduction in potentially beneficial bacterial species, primarily in women which may be explored for therapeutic potential.

Background: Whilst there is emerging evidence that the associations of the gut microbiota (GM) with hypertension differ between sexes, it remains uncertain whether the GM 1) is altered with antihypertensive-use, 2) can predict blood pressure (BP) trajectories, and 3) whether these associations are also sex-dependent. We therefore aimed to determine the sex-dependent associations of the GM with baseline hypertensive state in untreated versus treated participants and BP changes over time. Methods: A total of 451 community-dwelling middle-aged Hong Kong Chinese without symptomatic cardiovascular diseases were recruited (50% men, 14% on antihypertensive agents, mean age, 54.6±6.5 years). Shotgun metagenomic sequencing of stool samples and 24-hour ambulatory blood pressure monitoring (ABPM) were performed. The 24hr-ABPM was repeated on 139 returned subjects after 4 years. Statistical analysis was conducted with 4 covariate models that include age, sex, menopause status, body mass index (BMI), smoking, fasting glucose, triglyceride, cholesterol, sodium intake, and fatty liver status. Results: Amongst the 389/451 subjects not on antihypertensive agents, 167/389 (42.9%) had newly diagnosed hypertension at baseline. Females drove the significantly different b-diversity of the GM composition ( p <0.01) between the normotensives and hypertensives in the baseline study. Multiple GM species were significantly associated with hypertension. In sex stratified analysis, Faecalimonas umbilicate was significantly enriched in hypertensive females while Roseburia sp Am16-25 and Eubacterium ramulus were enriched in hypertensive males (all p <0.05-0.01). When comparing the 62 treated and 167 nontreated hypertensive subjects, the GM abundance, a-, and b-diversity were not significantly different. However, there were significant GM species associated with the use of antihypertensive medication. Notably, Lachnospiraceae bacterium and Flavonifractor plautii were significantly enriched in treated and untreated individuals, respectively (all p <0.05-0.001). During a mean follow up of 51.2±4.5 months, 73.2% of the 71 untreated subjects remained normotensive and were associated with enriched Bacteroides uniformis in their baseline metagenomic data under models adjusted for age, sex, and BMI. Conclusion: The GM displayed sex-dependent associations with hypertension within untreated and treated individuals and can potentially predict BP trajectory changes within a Chinese population.

Background: Short-term fluctuations in blood pressure (BP) as measured by 24-hr ambulatory BP monitoring (ABPM) is associated with subclinical atherosclerosis in the general population. However, whether short-term blood pressure variability (BPV) in the day versus night-time has differential effects on the development of subclinical atherosclerosis is uncertain. Methods and Results: We investigated the relationship between mean 24-hr BP and BPV parameters with common carotid artery intima-media thickness (CIMT) in 305 middle-aged Chinese (mean age 54±7years, 45% male) free from hypertension, hyperlipidaemia, diabetes or cardiovascular diseases. Subclinical atherosclerosis with elevated CIMT was defined as >75th percentile of CIMT of the overall study cohort. Linear regression models adjusted for age, sex, body mass index and smoking status revealed significant associations between office, day- and night-time mean SBP, day-time SBP standard deviation (SD) and coefficient of variation (CV) with elevated CIMT (all p<0.05). As shown in the Table , logistic regression analysis demonstrated that only ABPM day-time mean SBP (odds ratio [OR]:2.54, 95% confidence interval [95%CI]:1.29-5.00), night-time mean SBP (OR:3.23, 95%CI:1.67-6.22), night-time mean DBP (OR:2.27, 95%CI:1.16-4.42), day-time SBP CV (OR:0.47, 95%CI:0.26-0.86) and night-time SBP SD (OR1.94, 1.05-3.58) predicted the occurrence of subclinical atherosclerosis (all p<0.05). Conclusions: In healthy middle-age individuals without known hypertension, mean day and night-time SBP as measured using ABPM provided the strongest positive predictive value for elevated CIMT. Interestingly, increase day-time SBP CV versus night-time SBP SD have opposite effects on CIMT, suggesting circadian changes in SBP might have distinct contributions to the development of subclinical atherosclerosis.

Cancer and autoimmune disease lead to illness by damaging and evading a patients natural defenses. Immunotherapy methods are designed to reclaim and bolster the immune system to treat disease. This requires both highly specific binding of therapeutic agents to target molecules and optimizing techniques to harness the effector functions of the immune system. We describe methods for both these requirements. These include cell based systems for improving attachment to targets by assaying binding of therapeutic antibodies to immune checkpoint receptors and displaying molecular targets to CAR- T cells. They also include assays to improve immune effector functions such as Antibody-dependent cell-mediated cytotoxicity (ADCC) through cell based assays of IgG Fc binding to FCgRs.

Autophagy, a highly regulated homeostatic degradative process, allows cells to reallocate nutrients from less important to more essential processes under extreme conditions of starvation. Autophagy also prevents the buildup of damaged proteins and organelles that cause chronic tissue damage and disease. Although a topic of great interest with involvement of multiple signaling pathways, there are limitations in real-time detection of the autophagic process. EMD Millipore has developed technologies where prepackaged, ready-to-use, high-titer lentiviral particles, "lentiviral biosensors," encoding GFP- or RFP-tagged proteins provide a convenient and robust solution for fluorescent imaging of cells undergoing autophagy. Compared to nonviral transfection methods, lentiviral transduction, in many cases, offers higher transfection efficiency and more homogeneous protein expression, particularly for traditionally hard-to-transfect primary cell types. Lentiviral biosensors are ideal for use with fixed and live cell fluorescent microscopy, and are nondisruptive towards cellular function. GFP- or RFP-protein localization matches well with antibody-based immunostaining and demonstrates altered patterns of expression upon treatment with modulators of cell function and phenotype. Lentiviral biosensors provide a broadly effective, convenient method for visualization of cell behavior under a variety of physiological and pathological treatment conditions, in both endpoint and real-time imaging modalities. In this study, we focus on lentiviral biosensors containing GFP-LC3 and RFP-LC3 to study the formation of autophagosomes.

Elucidation of cellular regulation or disregulation can be monitored through a cells RNA expression levels. The ability to monitor gene expression within living cells that have become cancerous could provide better insight than examining fixed or lysed tissues when studying the complexities of cancerous biomarkers. However, monitoring RNA within intact cells can prove to be challenging with current techniques due to complex or harmful sample preparation techniques. Further, amplification methods can create false positives or erroneously inflate differences. Changes in RNA levels overtime are also impossible to detect without the use of duplicate samples. Here we describe the ability to detect specific RNA targets within living cells without the need for sample prep in real time. This technique also allows for the unique ability to reuse the cells following detection for additional experiments.

Abstract When studying the complexities of cancer biomarkers, the ability to monitor gene expression within live cells that have become cancerous provides better insight than examining fixed or lysed tissues. However, monitoring RNA within intact cells can prove to be challenging with current techniques, which involve complex RNA isolation procedures that compromise cell viability. Further, amplification methods can create false positives or erroneously inflate differences. Here we describe a probe-based detection method that allows for the detection of ErbB-2 and EGFR mRNA levels across live intact SK-BR-3 breast cancer cells without the need for transfection reagents. The detection methodology is based upon an oligonucleotide gold nanoparticle conjugate capable of detecting intracellular levels of mRNA and miRNA in living cells. The probes enter the cell by means of the cell's native endocytosis machinery. Upon binding to the complementary target RNA sequence, a fluorescent signal (flare) is released, which allows the cells to be detected on any fluorescence analysis platform. In contrast to traditional RNA detection methods, since our technique allows for detection within live cells without altering their gene expression, the same cells that were profiled for EGFR and ErbB-2 expression could be sorted based on their specific mRNA content and subsequently further studied. Stimulation or treatments could then be performed on the previously profiled cells and assessed using probes for additional RNA markers or more traditional techniques such as antibody staining, qRT-PCR, or Array profiling. For this purpose, we combined immunostaining for cell surface EGFR with fluorogenic nanoparticle-based detection of EGFR mRNA in 4 breast cancer cell lines known to range in EGFR expression levels, and observed significant correlation for the signals in two-color flow cytometry. Thus, detecting RNA expression levels in live cancer cells with the ability to utilize the same cells in downstream testing gives researchers the ability to perform experiments which were previously thought to be impossible. Citation Format: Don Weldon, Kevin Su, Alex Ko, Grace Johnston, Matthew Hsu, Lucas Armstrong. A novel approach to detecting RNA expression in living breast cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr LB-240. doi:10.1158/1538-7445.AM2013-LB-240

Cellular differentiation is a fundamental process in developmental biology. Progenitor cells must have the ability to differentiate into more specialized cell types for the body to respond to infections during autoimmunity. In an immunological response to infections, CD4+ T cells can give rise to a variety of T helper cells depending on the nature of the immune response, and subsequently release a distinct subset of signature cytokines. Similarly, when monocytes are exposed to established lineage specific conditions in vitro, human monocytes differentiate toward mature dendritic cells. In all cases, a characteristic inherent in cell differentiation is that during this process cells undergo a dramatic change in cell size, metabolic activity, and responsiveness to signals. Current methods such as flow cytometry or phase contrast morphology for determining cell differentiation are informative; however, drawbacks to these methods are that either it is too subjective or labor intensive. Here we utilized the coulter principle of impedance based particle detection as an alternative method for rapidly assessing cell differentiation. This study outlines a method for implementing the coulter principle to rapidly analyze CD4+ T cell differentiation towards various Th cell lineages, as well as monocyte differentiaton towards DCs. We have employed impedance based technology for determining cell volume to investigate the relationship between cell differentiation and cell size changes.

Abstract Assessment of autophagosome formation has been greatly facilitated by monitoring redistribution of LC3-fluorescent protein fusions. The sensitivity of the technique and capability for real-time analysis of autophagosome formation are advantageous over detection of endogenous LC3 redistribution in many experimental settings. However, the necessity for transfection can be an impediment to analysis of autophagosome formation in difficult-to-transfect cells, particularly primary cultures. We have developed lentiviral vectors (employing 3rd generation technology for optimal biosafety) encoding monomeric TagGFP2 and TagRFP fused at the N-termini of wild-type LC3 and LC3G120A as tools for analysis of autophagy in a wide variety of cell types. In immortalized cell lines and in primary cells (HUVEC and human mesenchymal stem cells) transduced with the GFP- and RFP-LC3 lentiviruses, amino acid deprivation in the presence of a lysosome inhibitor induced redistribution of the fluorescence from a diffuse cytoplasmic pattern to a punctate distribution. Minimal background of punctae in fed cells was observed. 3-Methyladenine blocked the starvation-induced redistribution, and the G120A mutant did not undergo detectable redistribution upon amino acid deprivation. In addition, we demonstrate effectiveness of lentiviral GFP-LC3 in HUVECs by flow cytometry, in which the plasma membrane is selectively permeabilized such that free cytosolic fluorescent protein-tagged LC3 is released while autophagosome-bound LC3 fusion protein is retained. We anticipate that these lentiviral autophagy biosensors will facilitate analysis of autophagosome formation in physiologically relevant primary cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4105. doi:1538-7445.AM2012-4105

Introduction Autophagy is an intracellular catabolic pathway which causes cellular protein and organelle turnover, and is associated with diverse diseases such as Alzheimer’s disease, cancer, and Crohn’s disease, in addition to aging. It is a tightly regulated process that plays a normal part in cell growth, development, and cellular homeostasis. Autophagy functions as a housekeeping mechanism through disposal of aging and dysfunctional proteins and organelles by sequestering and priming them for lysosomal degradation (Figure 1). Increasing evidence suggests that not only apoptosis, but also autophagy, can contribute to cell death and greatly influence general cell health. Malfunctions of autophagy can adversely impact longevity and the capability of cells to function at full capacity. In cancer cells, autophagy can compensate for hypoxic conditions and nutrient starvation; on the other hand, activation of cell death via autophagy can kill tumor cells. As a result, there is great interest in assays that can efficiently screen for activators and inhibitors of autophagy.

Naïve CD4+ T cells can give rise to a variety of subsets of Th cells depending on the nature of the immune response, and subsequently release a distinct subset of cytokines. Conventionally these cytokines can be measured in a secreted format (e.g. Luminex or ELISAs), but here we describe a novel method to reliably differentiate mouse CD4+ T cells, and further characterize the differentiated lineages by measuring intracellular cytokines using flow cytometry. In addition, we also describe the use of a proprietary fixable viability dye in order to eliminate the false positives associated with non-specific staining. Based on an optimized protocol for differentiating CD4+ T cells, we have developed differentiation tools designed to obtain the desired Th cell lineages. Following naïve T cell differentiation toward specific Th lineages, we further developed a flow cytometry assay to measure intracellular cytokine production by blocking the secretion of the cytokine with Brefeldin A. We further eliminate non-specific staining due to dead or dying cells using a proprietary fixable viability dye and accurately determine the percentage of Th lineages using guava benchtop flow cytometer and InCyte analysis software.

Detecting gene expression has traditionally been limited to technologies that examine expression in lysed or fixed cell populations. The ability to detect gene expression in live cells would allow for more physiologically relevant information based on a cell's response to given stimuli. Determining which genes were up or down regulated in those cells provides insight into complex gene regulatory networks and cell function. Here we present a novel RNA expression detection technology capable of detecting specific mRNA and miRNA in live, intact cells. This technology allows for carrier‐free cellular uptake of the reagent, followed by detection of target RNA, with the ability to perform downstream analysis in the same sample. This reagent has no toxic effects on cells, and we have demonstrated the ability to isolate and further propagate live cell populations based on gene expression level. This would allow researchers to detect RNA and subsequently protein levels within the same cell.Since current methods to interrogate the cellular RNA examine non‐native, amplified RNA targets, the relevance of such data often does not correlate to in vivo results. Furthermore, these methods do not provide RNA information from individual cells, and cells probed by these methods cannot be further studied following detection. Our technology presented here leaves the cells unchanged following detection, and therefore this same cell population can be used for downstream experiments. This research was funded by EMD Millipore.

The study of CD4+ T-helper (TH) cell differentiation is an important area of research that will aid in the understanding of inflammation and autoimmunity. CD4 + T cells can give rise to many subtypes depending on type of immune response. This study focuses on the TH1, TH2 and TH17 CD4+ T cell subtypes. Each subtype expresses a signature cytokine that directs the type of immune response needed. In order to analyze cytokine expression in TH cultures by flow cytometry, we employed a fixable viability dye to gate out dead cells that can accumulate in long-term differentiation cultures. In addition, flow cytometric analysis provides cell-specific information not obtained by ELISA analysis. Using standard TH culture protocols we differentiated naïve CD4+ T cells into TH1, TH2, and TH17 subtypes. We first stained the cells with the viability dye and then stained with antibodies against the signature cytokine of interest in less than 4 hours. Our data shows that we can easily obtain viability information while simultaneously evaluating cytokine production within our TH culture system. Using the fixable viability dye, we can exclude false positive cytokine staining and therefore obtain more accurate and reproducible intracellular expression data from cultured CD4+ T cells.

In der vorgestellten Studie werden Arzneimittel-induzierte DNA-Schäden und deren Wirkung im Zellzyklus mittels Durchflusszytometrie bestimmt. Somit kann Durchflusszytometrie eine Hochdurchsatz-Plattform für das Screening niedermolekularer Krebsmedikamente darstellen.