Flow Cytometry-based Quantification Research Articles

The short-term and long-term effects of bariatric/metabolic surgery on subcutaneous adipose tissue inflammation in humans

ContextThe mechanisms mediating the short- and long-term improvements in glucose homeostasis following bariatric/metabolic surgery remain incompletely understood. ObjectiveTo investigate whether a reduction in adipose tissue inflammation plays a role in the metabolic improvements seen after bariatric/metabolic surgery, both in the short-term and longer-term. DesignFasting blood and subcutaneous abdominal adipose tissue were obtained before (n=14), at one month (n=9), and 6–12months (n=14) after bariatric/metabolic surgery from individuals with obesity who were not on insulin or anti-diabetes medication. Adipose tissue inflammation was assessed by a combination of whole-tissue gene expression and flow cytometry-based quantification of tissue leukocytes. ResultsOne month after surgery, body weight was reduced by 13.5±4.4kg (p<0.001), with improvements in glucose tolerance reflected by a decrease in area-under-the-curve (AUC) glucose in 3-h oral glucose tolerance tests (−105±98mmol/L * min; p=0.009) and enhanced pancreatic β-cell function (insulinogenic index: +0.8±0.9pmol/mmol; p=0.032), but no change in estimated insulin sensitivity (Matsuda insulin sensitivity index [ISI]; p=0.720). Furthermore, although biomarkers of systemic inflammation and pro-inflammatory gene expression in adipose tissue remained unchanged, the number of neutrophils increased in adipose tissue 15–20 fold (p<0.001), with less substantial increases in other leukocyte populations. By the 6–12month follow-up visit, body weight was reduced by 34.8±10.8kg (p<0.001) relative to baseline, and glucose tolerance was further improved (AUC glucose −276±229; p<0.001) along with estimated insulin sensitivity (Matsuda ISI: +4.6±3.2; p<0.001). In addition, improvements in systemic inflammation were reflected by reductions in circulating C-reactive protein (CRP; −2.0±5.3mg/dL; p=0.002), and increased serum adiponectin (+1358±1406pg/mL; p=0.003). However, leukocyte infiltration of adipose tissue remained elevated relative to baseline, with pro-inflammatory cytokine mRNA expression unchanged, while adiponectin mRNA expression trended downward (p=0.069). ConclusionBoth the short- and longer-term metabolic improvements following bariatric/metabolic surgery occur without significant reductions in measures of adipose tissue inflammation, as assessed by measuring the expression of genes encoding key mediators of inflammation and by flow cytometric immunophenotyping and quantification of adipose tissue leukocytes.

Accurate Quantification of T Cells by Measuring Loss of Germline T-Cell Receptor Loci with Generic Single Duplex Droplet Digital PCR Assays

Quantifying T cells accurately in a variety of tissues of benign, inflammatory, or malignant origin can be of great importance in a variety of clinical applications. Flow cytometry and immunohistochemistry are considered to be gold-standard methods for T-cell quantification. However, these methods require fresh, frozen, or fixated cells and tissue of a certain quality. In addition, conventional and droplet digital PCR (ddPCR), whether followed by deep sequencing techniques, have been used to elucidate T-cell content by focusing on rearranged T-cell receptor (TCR) genes. These approaches typically target the whole TCR repertoire, thereby supplying additional information about TCR use. We alternatively developed and validated two novel generic single duplex ddPCR assays to quantify T cells accurately by measuring loss of specific germline TCR loci and compared them with flow cytometry-based quantification. These assays target sequences between the Dδ2 and Dδ3 genes (TRD locus) and Dβ1 and Jβ1.1 genes (TRB locus) that become deleted systematically early during lymphoid differentiation. Because these ddPCR assays require small amounts of DNA instead of freshly isolated, frozen, or fixated material, initially unanalyzable (scarce) specimens can be assayed from now on, supplying valuable information about T-cell content. Our ddPCR method provides a novel and sensitive way for quantifying T cells relatively fast, accurate, and independent of the cellular context.

Improvements in glycemic control after gastric bypass occur despite persistent adipose tissue inflammation.

Type 2 diabetes commonly goes into remission following Roux-en-Y gastric bypass (RYGB). As the mechanisms remain incompletely understood, a reduction in adipose tissue inflammation may contribute to these metabolic improvements. Therefore, whether RYGB reduces adipose tissue inflammation compared with equivalent weight loss from an intensive lifestyle intervention was investigated. Sixteen people with obesity and type 2 diabetes were randomized to RYGB or lifestyle intervention. Fasting blood and subcutaneous abdominal adipose tissue were obtained before and after the loss of ∼7% of baseline weight. Adipose tissue inflammation was assessed by whole-tissue gene expression and flow cytometry-based quantification of tissue leukocytes. At 7% weight loss, insulin and metformin use were reduced among the RYGB but not the Lifestyle cohort, while fasting glucose and insulin declined in both. Adipose tissue inflammation increased modestly after RYGB and to a similar extent following nonsurgical weight loss. In both groups, the number of neutrophils increased severalfold (P < 0.001), mRNA levels of the proinflammatory cytokine interleukin-1β increased (P = 0.037), and mRNA expression of the anti-inflammatory and insulin-sensitizing adipokine adiponectin decreased (P = 0.010). A reduction in adipose tissue inflammation is not one of the acute weight loss-independent mechanisms through which RYGB exerts its antidiabetes effects.

Validation of a flow cytometry-based detection of γ-H2AX, to measure DNA damage for clinical applications.

The nucleosomal histone protein H2AX is specifically phosphorylated (γ-H2AX) adjacent to DNA double-strand breaks (DSBs) and is used for quantifying DSBs. Many chemotherapies and ionizing radiation (IR) used in cancer treatment result in DSBs. Therefore, γ-H2AX has a significant potential as a biomarker in evaluating patient sensitivity and responsiveness to IR and chemotherapy. Here, we report a flow cytometry-based quantification of γ-H2AX (FCM-γ-H2AX assay) customized for clinical practice. We validated that our method is able to detect DNA damage in peripheral blood mononuclear cells (PBMCs) treated with DSB inducing agents. The method also detected the DNA repair deficiency in PBMCs treated with DNA repair inhibitors, as well as the deficiency in DNA repair signaling in PBMCs from two ataxia telangiectasia patients. The FCM-γ-H2AX assay has sufficient analytical sensitivity and precision to measure levels of DNA damage and DNA repair for clinical purposes. © 2016 International Clinical Cytometry Society.

Flow Cytometry-Based Quantification of HIV-Induced T Cell Chemotactic Response.

The human immunodeficiency virus (HIV) infects blood CD4 T cells through binding to CD4 and the chemokine co-receptor, CXCR4 or CCR5. This viral binding to CXCR4 or CCR5 also triggers the activation of a variety of signaling molecules such as LIMK/cofilin and WAVE2/Arp2/3 to promote actin dynamics, which are necessary for viral nuclear migration and the latent infection of blood resting CD4 T cells. In this chapter, we describe the methods for quantification of HIV-induced actin polymerization and cofilin phosphorylation in human T cells using flow cytometry.

Prerequisites for the analysis and sorting of extracellular vesicle subpopulations by high-resolution flow cytometry.

Submicron-sized vesicles released by cells are increasingly recognized for their role in intercellular communication and as biomarkers of disease. Methods for high-throughput, multi-parameter analysis of such extracellular vesicles (EVs) are crucial to further investigate their diversity and function. We recently developed a high-resolution flow cytometry-based method (using a modified BD Influx) for quantitative and qualitative analysis of EVs. The fact that the majority of EVs is <200 nm in size requires special attention with relation to specific conditions of the flow cytometer, as well as sample concentration and event rate. In this study, we investigated how (too) high particle concentrations affect high-resolution flow cytometry-based particle quantification and characterization. Increasing concentrations of submicron-sized particles (beads, liposomes, and EVs) were measured to identify coincidence and swarm effects, caused by the concurrent presence of multiple particles in the measuring spot. As a result, we demonstrate that analysis of highly concentrated samples resulted in an underestimation of the number of particles and an interdependent overestimation of light scattering and fluorescence signals. On the basis of this knowledge, and by varying nozzle size and sheath pressure, we developed a strategy for high-resolution flow cytometric sorting of submicron-sized particles. Using the adapted sort settings, subsets of EVs differentially labeled with two fluorescent antibodies could be sorted to high purity. Moreover, sufficient numbers of EVs could be sorted for subsequent analysis by western blotting. In conclusion, swarm effects that occur when measuring high particle concentrations severely hamper EV quantification and characterization. These effects can be easily overlooked without including proper controls (e.g., sample dilution series) or tools (e.g., oscilloscope). Providing that the event rate is well controlled, the sorting strategy we propose here indicates that high-resolution flow cytometric sorting of different EV subsets is feasible.

The Role of Rac and Pak in Neutrophil Histone Hypercitrullination and Neutrophil Extracellular Traps Formation

Under specific activating conditions, polymorphonuclear neutrophils (PMNs) release neutrophil extracellular traps (NETs) composed of decondensed chromatin lined with microbicidal protein such as neutrophil elastase and myeloperoxidase. NETs contribute to innate immunity but can also foster autoimmune diseases and thrombus formation. NET formation (NETosis) requires reactive oxygen species (ROS) production by NADPH oxidase and histone hypercitrullination by peptidylarginine deiminase 4 (PAD4), allowing for chromatin decondensation. Rac GTPases are expressed in three isoforms: Rac1 is ubiquitously expressed and plays a role in PMN migration and oxidase function; Rac2 is hematopoietic-specific and the major isoform in PMNs and Rac3 is mostly neuronal. Rac1 and Rac2 regulate the cytoskeleton in PMNs, controlling actin polymerization, cell shape, adhesion and migration and are essential components of the NADPH oxidase complex. The present study aimed to explore the role of the Rac pathway on NETosis in PMNs, including the upstream guanosine exchange factor (GEF) activator, Vav, and a downstream effector of Rac, p21 activated kinase, Pak. We developed a flow cytometry-based quantification of H3 hypercitrullination (H3Cit). In response to phorbol myristate ester (PMA) stimulation, H3Cit is increased to 136% of basal in WT cells, compared with 103% in Rac2-/- (P<0.01) (Table). H3Cit levels observed by flow were confirmed in a NET formation assay. Rac2-/- PMNs formed significantly fewer NETs both spontaneously and after PMA stimulation (WT unstimulated 2.79%, Rac2-/- unstimulated 0.72%, WT+PMA 10.84%, Rac2-/-+PMA 1.39%, P< 0.05 for all pair comparisons). Furthermore, Rac2-/- mice demonstrated a trend towards reduced frequency of provoked thrombosis in an in vivo vena cava stenosis model (WT 78% and Rac2-/- 56% of mice with thrombus). Deletion of floxed Rac1 sequences in a Rac2-/- background in vivoallows generation and purification of PMNs lacking both Rac isoforms. Rac1Δ/Δ,Rac2-/- PMNs, which are defective in actin polymerization, had reduced basal H3Cit and a nearly complete lack of PMA-induced increase in H3Cit (136% vs 69%, WT vs Rac1Δ/Δ,Rac2-/-, P<0.01) (Table). Null knockouts of the GEFs Vav1 (hematopoietic-specific), Vav2, Vav3 or both Vav1 and 3 did not impair H3Cit response to PMA (Table). We next studied downstream effectors of Rac. Group A Paks include Pak1, 2 and 3 isoforms. Pretreatment of wild-type PMNs with either PF3758309 or IPA-3, two group A Pak inhibitors with distinct mechanisms of action, led to reduced H3Cit after PMA stimulation (induction reduced from 36% to 11% for both PF3758309 and IPA-3 treated (Table). To validate this in a genetic model, we studied Pak2Δ/Δ PMNs, since we have recently demonstrated the dependence of hematopoietic stem cell migration on Pak2. Pak2Δ/Δ demonstrated a reduced basal level of H3Cit and a significantly reduced PMA-induced increase in H3Cit (136% vs 94%, WT vs Pak2Δ/Δ, P<0.05, Table). In summary, we describe a flow-based assay that quantitates the early processes of NET formation and validated that this assay reliably predicts agonist-induced NET formation in a genetic model. The results establish that both Rac1 and Rac2, and the downstream effector Pak2, regulate histone H3 hypercitrullination and NET formation in PMNs, while suggesting that Vav does not activate the Rac pathway in PMA-induced NET formation. These data further delineate the role of the Rac pathway in NETosis, linking cytoskeleton and oxidase functions. Furthermore, these data indicate Pak could represent a therapeutic target for a wide array of pathological processes related to NETosis such as thrombosis and numerous autoimmune diseases.TableIntensity of H3Cit staining as determined by flow cytometry-based assay.Basal H3Cit levelPMA-induced H3CitPMA-induced changeWT PMN100±2%136±5%36%Rac2-/-82±9% ns103±15.3%**21%Rac1Δ/Δ, Rac2-/-64±10%***69±10%**5%Vav1-/-86±9% ns145±15% ns59%Vav2-/-91±4% ns134±18% ns43%Vav3-/-153±30%*171±28% ns18%Vav1,3-/-125±20% ns144±24% ns19%WT+PF 5nM76±17% ns87±8%*11%WT+ IPA 5µM100±4% ns111±10% ns11%Pak2Δ/Δ75±7%*94±11%*19%Results are expressed as mean±SEM % of the untreated WT control of each experiment. Results are from ≥3 independent experiments. * P<0.05, **P<0.01. P<0.001, ns non-significant, by two-tailed t-test. DisclosuresNo relevant conflicts of interest to declare.

Identification and quantification of basophils in the airways of asthmatics following segmental allergen challenge.

During asthma attacks, allergens activate sensitized basophils in the lung, thereby aggravating symptoms. Due to the paucity of basophils in bronchial lavage fluid and the lack of specific basophil detection and quantification methods, basophil-directed research in these samples was hampered in the past. This study aimed to establish and validate a flow cytometry-based basophil detection and quantification method for human basophils from bronchoalveolar lavage (BAL) and blood as a prerequisite for a better understanding of their pathogenic contribution and subtyping of asthma phenotypes. BAL and blood leukocytes from seasonal asthmatics were analyzed by flow cytometry. Chipcytometry, a highly sensitive single-cell analysis method, was used to validate the staining panel for basophils. Cell differentials of May-Grünwald-Giemsa-stained cytospins were used to compare basophil percentages. BAL basophils are identifiable as CD123(+) HLA-DR(-) CD3(-) CD14(-) CD19(-) CD20(-) CD56(-) cells in flow cytometrical analysis. Their identity was validated by Chipcytometry. CD203c was highly expressed by BAL basophils, whereas it was expressed at variable levels on blood basophils. The two quantification methods correlated, although more basophils were detected by flow cytometry. Furthermore, the increase in basophil percentages in the lung correlated with the decrease in the basophil percentages in the blood after allergen challenge. We here validated a reliable basophil quantification method, which is independent of the cell's activation and degranulation state. The results obtained with this method indicate that basophils are directly recruited from the blood circulation to the airway lumen.

Improved Igh-Based MRD Detection By Using Droplet Digital PCR: a Comparison With Real Time Quantitative PCR In MCL and MM

BackgroundIn mature lymphoid disorders, minimal residual disease (MRD) detection based on real time quantitative PCR (RQ-PCR) of immunoglobulin heavy chain gene rearrangement (IgH) has a well-established role in prognostic assessment, particularly in Mantle cell Lymphoma (MCL) and Multiple Myeloma (MM). RQ-PCR has excellent sensitivity and specificity but has a major limitation in its relative quantification nature, as it requires a reference standard curve usually built with dilutions of diagnostic tumor DNA or on plasmids containing the target rearrangement. Droplet Digital PCR (DD-PCR), applying the principle of limiting dilution of DNA and single molecule detection allows a reliable absolute quantification of target. In this study we compared IgH-based MRD detection by RQ-PCR and DD-PCR, to assess whether DD-PCR could achieve the same performances of RQ-PCR in the absence of the limitation mentioned above. MethodsBone marrow (BM) and peripheral blood (PB) samples were collected from patients affected by MCL and MM in which RQ-PCR based MRD analysis was already performed in the context of prospective clinical trials. In all trials patients gave the informed consent for MRD determination. IgH-based MRD detection by RQ-PCR was carried out as previously described [Ladetto et al. BBMT 2000] and results were interpreted according to the Euro-MRD guidelines [van der Velden et al. Leukemia 2007]. DD-PCR was performed by the QX100 Droplet Digital PCR system (Bio-RAD Inc.) on 500 ng of genomic DNA combined with the same Allele Specific Oligonucleotides (ASO)-primers and TaqMan-probes used in the RQ-PCR. Droplets were generated by QX100 droplet generator. End-point PCR (40 cycles) was performed on a T100 Thermal cycler (Bio-RAD Inc). The PCR product was loaded in the QX100 droplet reader and analyzed by QuantaSoft 1.2 (Bio-Rad Inc). For data interpretation RQ-PCR and DD-PCR results were expressed as amount of target copies per 1E+05 cells. Comparability of MRD results by DD-PCR and RQ-PCR was assessed by means of bivariate correlations between methods analysis (R2.15.1 package irr). Discordances were classified as follows: a positive/negative discordance was defined as major when the positive result was >1E-04 and minor when ≤1E-04; a quantitative discordance was defined as the presence of two positive results with a quantitative discrepancy >1 log. ResultsOverall, 161 samples belonging to 35 patients (18 MCL and 17 MM), 66 MCL and 95 MM were analyzed. 35 samples were taken at diagnosis and 126 at follow-up. 118 were BM while 43 were PB. A significant correlation was found between DD-PCR and RQ-PCR (R2=0.89, p<0.0001) (fig). DD-PCR and RQ-PCR showed superimposable sensitivity (10-5). Specificity in terms of appearance of non-specific amplifications signals in no-template samples (tested for all patients) and reproducibility on 30 replicates (4 samples) were superimposable. 128 out of 161 samples were fully concordant (Choen's K=0.80). MRD detection was concordantly positive in 106/161 (65.8%) samples and concordantly negative in 22/161 samples (13.7%). Only 5/161 (3.1%) samples showed major qualitative discordance. 28/161 (17.4%) samples showed minor qualitative discordance (which might be related to Poisson's statistics). Quantitative discordances were observed in 5/161 (3.1%) of cases (positive non quantifiable (PNQ) cases were conventionally placed to a value intermediate between sensitivity and quantitative range). Interestingly, 17 samples negative by RQ-PCR were scored positive by DD-PCR (median 6 copies, range 2-74) while 16 samples positive by RQ-PCR (median 5 copies, range 2-44) were negative by DD-PCR. ConclusionsHere we report for the first time the use of DD-PCR in the context of IgH-based MRD evaluation in lymphoproliferative disorders. DD-PCR is a feasible tool for IGH-based MRD monitoring in MCL and MM, reaching similar sensitivities compared to standardized RQ-PCR. Moreover DD-PCR allows bypassing the need of building a standard curve thus considerably reducing the complexity of IgH-based RQ-PCR (need of purified diagnostic tissue or Flow Cytometry-based quantification of tumor load or diagnosis, or building of a plasmid-derived standard curve). Finally DD-PCR might potentially overcome the problem of positive non-quantifiable samples. These features make DD-PCR a feasible and attractive alternative method for IgH-based MRD assessment. [Display omitted] Disclosures:Kubiczkovà:GAP304/10/1395 : Research Funding; MUNI/11/InGA17/2012: Research Funding.

Flow cytometry-based quantification of cell proliferation in the mixed cell co-culture.

In cell communities, among the crucial signals that govern cell function are those generated locally by surrounding cells. A co-culture of mixed homotypic or heterotypic cells, which is often used in various fields of experimental biology and medicine, can be applied for elucidation of the role of cell proximity in modulating proliferative responses. Quick and reliable quantification of the changes in proliferation of each of the mixed cell populations as a result of their co-culture is of importance. For this purpose, flow cytometry together with fluorescent tracers that do not affect cell proliferation can be used.

Abstract 5340: Standardization of cancer stem cell enumeration and isolation methods allows cross comparison among different tumor entities

Abstract Growing knowledge concerning tumor heterogeneity has increased the importance of analyzing tumor resident cell and stem cell subpopulations. In particular, cancer stem cells (CSCs), also called tumor initiating cells, have gained substantial interest in the research field over the past few years. CSCs have been isolated from multiple tumor entities and were shown to play a crucial role during tumor growth and metastasis. However, isolation and analysis of CSCs even from single tumor entities like melanoma showed contradictory results among different research groups. This can at least partially be explained by variations in the dissociation and isolation methods used. One critical point that has to be considered during the analysis of putative CSC markers is that the procedure used for dissociation of primary tissue prior to staining may cause a technical bias. Aggressive proteases, such as trypsin, which cleave off several CSC surface markers like ABCB5 or CD44, may cause incorrect results concerning the differential expression of sensitive markers (Civenni et al., 2011). A further source of non-reproducible findings is the use of antibodies that are specific to sub-forms of cell surface markers. Glycosylation and splice-isoform dependent epitopes, as found for e.g. CD24 and CD133, are differentially expressed among tissues and cell states. In particular, it was shown that the AC133 epitope but not the entire CD133 protein expression is lost upon CSC differentiation (Kemper et al., 2010). We have developed a standardized platform that allows fast and highly reproducible CSC enumeration and isolation from various tumor tissues. This platform includes automated and therefore user independent procedures for dissociation of human and implanted mouse tumor tissue without degradation of cell surface epitopes yielding high amounts of single cells at viabilities around 90%. Furthermore, antibody conjugates of clones recognizing epitopes shown to be relevant for CSC analysis were generated and tested on cell lines as well as primary tumor tissue allowing for the quantification and isolation of CSCs. As a proof of principle, highly viable populations of CD133+ and CD45−/CD24−/CD44+ CSCs were efficiently isolated from a glioblastoma at 86% and an invasive ductal mamma carcinoma at 94% purity, respectively. In addition, optimized gating and exclusion strategies are presented avoiding analytical bias for flow cytometry-based CSC quantification from primary tissue as shown for human melanoma tissue. By using reliable methods for the analysis of CSCs from various origins, a major cause of conflicting results in this field may be minimized and comparisons of these subpopulations among distinct tumor entities will be possible. 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 5340. doi:1538-7445.AM2012-5340

Regulation of monocyte subset proinflammatory responses within the lung microvasculature by the p38 MAPK/MK2 pathway

Margination and activation of monocytes within the pulmonary microcirculation contribute substantially to the development of acute lung injury in mice. The enhanced LPS-induced TNF expression exhibited by Gr-1high compared with Gr-1low monocytes within the lung microvasculature suggests differential roles for these subsets. We investigated the mechanisms responsible for such heterogeneity of lung-marginated monocyte proinflammatory response using a combined in vitro and in vivo approach. The monocyte subset inflammatory response was studied in vitro in mouse peripheral blood mononuclear cell-lung endothelial cell coculture and in vivo in a two-hit model of intravenous LPS-induced monocyte margination and lung inflammation in mice, by flow cytometry-based quantification of proinflammatory genes and intracellular phospho-kinases. With LPS stimulation in vitro, TNF expression was consistently higher in Gr-1high than Gr-1low monocytes, markedly enhanced by coculture with endothelial cells, and abrogated by p38 MAPK inhibitors. Expression of IL-6, inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) was only detectable under coculture conditions, was substantially higher in Gr-1high monocytes, and was attenuated by p38 inhibition. Consistent with these differential responses, phosphorylation of p38 and its substrate MAPK-activated protein kinase 2 (MK2) was significantly higher in the Gr-1high subset. In vivo, p38 inhibitor treatment significantly attenuated LPS-induced TNF expression in “lung-marginated” Gr-1high monocytes. LPS-induced p38/MK2 phosphorylation was higher in lung-marginated Gr-1high than Gr-1low monocytes and neutrophils, mirroring TNF expression. These results indicate that the p38/MK2 pathway is a critical determinant of elevated Gr-1high subset responsiveness within the lung microvasculature, producing a coordinated proinflammatory response that places Gr-1high monocytes as key orchestrators of pulmonary microvascular inflammation and injury.

P.2.e.006 Clinical features of early onset bipolar disorder

The mechanisms mediating the short- and long-term improvements in glucose homeostasis following bariatric/metabolic surgery remain incompletely understood.To investigate whether a reduction in adipose tissue inflammation plays a role in the metabolic improvements seen after bariatric/metabolic surgery, both in the short-term and longer-term.Fasting blood and subcutaneous abdominal adipose tissue were obtained before (n = 14), at one month (n = 9), and 6–12 months (n = 14) after bariatric/metabolic surgery from individuals with obesity who were not on insulin or anti-diabetes medication. Adipose tissue inflammation was assessed by a combination of whole-tissue gene expression and flow cytometry-based quantification of tissue leukocytes.One month after surgery, body weight was reduced by 13.5 ± 4.4 kg (p < 0.001), with improvements in glucose tolerance reflected by a decrease in area-under-the-curve (AUC) glucose in 3-h oral glucose tolerance tests (− 105 ± 98 mmol/L * min; p = 0.009) and enhanced pancreatic β-cell function (insulinogenic index: + 0.8 ± 0.9 pmol/mmol; p = 0.032), but no change in estimated insulin sensitivity (Matsuda insulin sensitivity index [ISI]; p = 0.720). Furthermore, although biomarkers of systemic inflammation and pro-inflammatory gene expression in adipose tissue remained unchanged, the number of neutrophils increased in adipose tissue 15–20 fold (p < 0.001), with less substantial increases in other leukocyte populations. By the 6–12 month follow-up visit, body weight was reduced by 34.8 ± 10.8 kg (p < 0.001) relative to baseline, and glucose tolerance was further improved (AUC glucose − 276 ± 229; p < 0.001) along with estimated insulin sensitivity (Matsuda ISI: + 4.6 ± 3.2; p < 0.001). In addition, improvements in systemic inflammation were reflected by reductions in circulating C-reactive protein (CRP; − 2.0 ± 5.3 mg/dL; p = 0.002), and increased serum adiponectin (+ 1358 ± 1406 pg/mL; p = 0.003). However, leukocyte infiltration of adipose tissue remained elevated relative to baseline, with pro-inflammatory cytokine mRNA expression unchanged, while adiponectin mRNA expression trended downward (p = 0.069).Both the short- and longer-term metabolic improvements following bariatric/metabolic surgery occur without significant reductions in measures of adipose tissue inflammation, as assessed by measuring the expression of genes encoding key mediators of inflammation and by flow cytometric immunophenotyping and quantification of adipose tissue leukocytes.

Extracts of Salvia officinalis from different growing areas and their antiviral effect against enveloped and non-enveloped viruses

In our study we compared extracts from Salvia officinalis L. (sage), which was grown at two different areas in Germany, for their antiviral effect. Firstly it was cultivated in an dry and hot area (area 1) and secondly in an area with a cooler climate and more rain (area 2). Different extracts were made from both samples with water/ethanol- mixtures in the ratio of 0, 20, 40, 60 and 80% ethanol, respectively. A HPLC analysis of the extracts showed as major components apigenin-7-glucuronide, luteolin- 3-glucuronide and rosmarinic acid. The antiviral effects of these extracts were tested against the enveloped herpes simplex virus type 1 (HSV-1) and the non-enveloped adenovirus type 5. In order to determine the mode of antiviral action, the extracts were added to the cells or viruses at different times during infection. The inhibitory activity against HSV-1 was tested using a plaque reduction assay. Adenoviral GFP reporter gene expression allowed for a flow cytometry-based quantification of infection levels. When HSV-1 was pretreated with the extracts prior to adsorption, plaque formation was reduced by >90%. The best results were shown by the extracts from area 1. In time-response studies over a period of 2 hours, a clearly time-dependent activity for all extracts was demonstrated. Already after 20 minutes of incubation of HSV-1 with the extracts, an antiviral activity of about 70–80% was shown. Pretreatment of adenovirus with the extracts for 1 hour showed an inhibitory effect on adenoviral infection from 20% (80% ethanol) up to 70% (20% ethanol). Therefore the extracts show antiviral activity independent of an envelope.

A reproducible method for the enumeration of functional (cytokine producing)versusnon-functional peptide-specific cytotoxic T lymphocytes in human peripheral blood

One of the most difficult laboratory challenges in the field of therapeutic cancer vaccines has been the development of uncomplicated/reproducible methods for the quantification of vaccine immunization efficacy in peripheral blood of cancer patients. Existing methods are limited by lack of functional information (tetramers), difficulties with standardization/reproducibility [enzyme-linked immunosorbent spot (ELISPOT)] and reliance on endogenous (sample-specific) antigen presentation (cytokine flow cytometry). Herein we present a reproducible method utilizing an artificial antigen-presenting cell platform for flow cytometry-based quantification of the frequency and activation status of peptide-specific cytotoxic T lymphocytes. The methodology [currently presented for cytomegalovirus human leucocyte antigen (HLA)-A2 cognant peptide antigens] allows simultaneous ex vivo quantification of activated (cytokine-producing) and inactive tetramer-positive T cells following HLA class I/peptide/CD28 stimulation independent of endogenous antigen presentation. The simplicity and reliability of the assay provide for high-throughput applications and automation. The utility and application of this method are discussed.

Quantification of bacterial invasion into adherent cells by flow cytometry

Quantification of invasive, intracellular bacteria is critical in many areas of cellular microbiology and immunology. We describe a novel and fast approach to determine invasion of bacterial pathogens in adherent cell types such as epithelial cells or fibroblasts based on flow cytometry. Using the CEACAM-mediated uptake of Opa-expressing Neisseria gonorrhoeae as a well-characterized model of bacterial invasion, we demonstrate that the flow cytometry-based method yields results comparable to a standard antibiotic protection assay. Furthermore, the quantification of intracellular bacteria by the novel approach is not biased by intracellular killing of the microbes and correctly discriminates between cell-associated extracellular and bona fide intracellular bacteria. As flow cytometry-based quantification is also applicable to other pathogen-host interactions such as the integrin-mediated internalization of Staphylococcus aureus, this approach provides a fast and convenient alternative for the quantification of bacterial uptake and should be particularly useful in elucidating the molecular mechanisms of pathogen-triggered host cell invasion.

Use of 5-Fold Staining for Multiparameter Flow Cytometry-Based Quantification of Minimal Residual Disease in Acute Myeloid Leukemia Results in Improved Sensitivity.

The quantification of minimal residual disease (MRD) by multiparameter flow cytometry (MFC) using triple staining has been shown to yield prognostic information independent of other parameters in patients with acute myeloid leukemia (AML). Due to the immunophenotypic heterogeneity of AML the application of 5-fold staining may result in a better characterization of the leukemia-associated aberrant immunophenotype (LAIP) and thus in an improved sensitivity of the method as compared to triple staining. We analyzed bone marrow samples from 114 patients with newly diagnosed and untreated AML by MFC using a comprehensive antibody panel with 5-fold combinations. Sensitivity was estimated by quantification of LAIP-positive cells for each LAIP in 18 normal bone marrow samples. In each patient at least one LAIP was identified (total, 203 LAIPs). The LAIPs were present on a median of 15.88% of the bone marrow cells at diagnosis (range, 2.11% to 79.64%). The median number of normal bone marrow cells displaying the LAIPs ranged from 0.001% to 0.065% (median, 0.010%). As a result, the logarithmic difference (LD) in LAIP-positive cells between leukemic and normal bone marrow amounted to a median of 3.33 (range, 1.96 to 4.88). Similarly, if only the most sensitive LAIP was considered for each patient the median frequencies of LAIP-positive cells were 14.07% (range, 2.11% to 77.57%) in leukemic bone marrow and 0.010% (range, 0.001% to 0.065%) in normal bone marrow. Importantly, however, in this setting the resulting LD amounted to a median of 3.45 (range, 1.96 to 4.88). In order to estimate the impact of applying 5-fold staining on the sensitivity the information of each of the applied colors was skipped once while the results of the other four colors, respectively, were used. Skipping one color resulted in an increase of LAIP-positive normal bone marrow cells (median, 0.050%; range, 0.001% to 3.6%) while the percentages of LAIP-positive leukemic cells changed only marginally (median, 22.65%; range, 2.25% to 90.06%). The gain in LD by applying 5-fold staining in comparison to 4-fold staining amounted to a median of 0.58 (maximum gain, 3.14). In 32 patients a total of 120 follow-up samples have been analyzed appyling the combination of antibodies that allowed the best LAIP definition. The LD from diagnosis to follow-up amounted to a median of 2.82 (range, 0.77 to 4.82). Clinical follow-up data is available in 26 of these 32 patients. MRD assessment after completion of consolidation therapy has been performed in 15 patients. The median LD between diagnosis and follow-up assessment is 2.84 (range, 1.07 to 4.33). Separating patients according to this median LD identified a group of patients with no relapses yet (LD >2.84) while patients with an LD <2.84 had an event-free survival of only 50% at one year (p=0.075). These data confirm that flow cytometrically-based assessment of MRD is feasible in AML and results in prognostic information. It is suggested that the application of 5-fold staining significantly improves the sensitivity and thereby the overall accuracy of the method.

Improved quantification of cell survival on stromal monolayers by flow cytometric analyses

The ex vivo survival of leukemic cells maintained on bone marrow stroma is an important tool for the investigation of cell survival and leukemogenesis. Currently, ex vivo survival of leukemic cell survival is measured by coculture on stromal cell monolayers. In these assays, we postulated that two important sources of error might be introduced through either variations in flow volume or in donor stromal cells. A previously reported coculture assay that maintains leukemic cells on bone marrow stromal cells was employed. We identified two means of optimizing the coculture assay. First, biologically inert beads having well-characterized fluorescent properties were added to each sample to mathematically adjust for flow-based variations in volume acquisition. The inclusion of fluorescent beads to the basic stromal cell assay showed a significantly lower coefficient of variation as compared to samples analyzed without beads or manually counted using a hemacytometer. Second, in order to minimize variability in bone marrow hematopoietic function between donors, an adherent stromal cell line known to support hematopoiesis (HS-5) was used. When normal human donor stromal cells were used, variability in the survival of leukemic cells was observed on stromal cells derived from different donors. In contrast, statistically significant variability in survival of leukemic cells was not seen on HS-5 monolayers. Finally, we demonstrate that patient-derived leukemic samples may be examined for cell survival using these modifications. The novel use of fluorescent beads and a hematopoietic-supportive stromal cell line together makes the quantification of stroma-supported cell survival more reproducible, accurate, and amenable to patient-derived samples. These improvements in flow cytometry-based cell quantification are an important step in establishing a role for stromal cell assays in the study of leukemia biology and therapy.