Monocyte subtype counts are associated with 10-year cardiovascular disease risk as determined by the Framingham Risk Score among subjects of the LIFE-Adult study.

Coronary heart disease, an inflammatory disease, is the leading cause of death globally. White blood cell counts (including monocytes) are easily available biomarkers of systemic inflammation. Monocyte subtypes can be measured by flow cytometry and classified into classical (CD14high, CD16neg), intermediate (CD14high, CD16+) and non-classical (CD14+, CD16high) with distinct functional properties. The goal of this study was to investigate the association of monocyte total count and its subtypes with cardiovascular risk groups defined by the Framingham Risk Score, which is used to estimate the 10-year risk of developing myocardial infarction or predict mortality following coronary heart disease. We also aimed to investigate whether monocyte counts are associated with relevant cardiovascular risk factors not included in the Framingham Risk Score, such as carotid atherosclerotic plaque and intima-media thickness. Our data came from the LIFE-Adult study, a population-based cohort study of 10,000 randomly selected participants in Leipzig, Germany. Data was gathered using self-administered questionnaires and physical examinations. Carotid plaques and intima-media thickness were measured using carotid artery sonography. Monocyte subtypes in blood were determined by 10-color flow cytometry for a total of 690 individuals. In a multivariate regression analysis adjusting for the risk factors BMI, intima-media thickness, presence of carotid plaques and diabetes mellitus, monocyte subtypes and total count were found to be significantly associated with the dichotomized Framingham Risk Score (≥10% versus <10%): Odds ratios [95% confidence interval] for monocyte subtypes: classical: 11.19 [3.79-34.26]; intermediate: 2.27 [1.11-4.71]; non-classical: 4.18 [1.75-10.20]; total: 14.59 [4.61-47.95]. In absence of prospective data, the FRS was used as a surrogate for CHD. Our results indicate that monocyte counts could provide useful predictive value for cardiovascular disease risk.

Discrimination of Head and Neck Squamous Cell Carcinoma Patients and Healthy Adults by 10-Color Flow Cytometry: Development of a Score Based on Leukocyte Subsets.

BACKGROUND: Leukocytes in peripheral blood (PB) are prognostic biomarkers in head and neck squamous cell carcinoma cancer patients (HNSCC-CPs), but differences between HNSCC-CPs and healthy adults (HAs) are insufficiently described. METHODS: 10-color flow cytometry (FCM) was used for in-depth immunophenotyping of PB samples of 963 HAs and 101 therapy-naïve HNSCC-CPs. Absolute (AbsCC) and relative cell counts (RelCC) of leukocyte subsets were determined. A training cohort (TC) of 43 HNSCC-CPs and 43 HAs, propensity score (PS)-matched according to age, sex, alcohol, and smoking, was used to develop a score consecutively approved in a validation cohort (VC). RESULTS: Differences in AbsCC were detected in leukocyte subsets (p < 0.001), but had low power in discriminating HNSCC-CPs and HAs. Consequently, RelCC of nine leukocyte subsets in the TC were used to calculate 36 ratios; receiver operating characteristic (ROC) curves defined optimum cut-off values. Binary classified data were combined in a score based on four ratios: monocytes-to-granulocytes (MGR), classical monocytes-to-monocytes (clMMR), monocytes-to-lymphocytes (MLR), and monocytes-to-T-lymphocytes (MTLR); ≥3 points accurately discriminate HNSCC-CPs and HAs in the PS-matched TC (p = 2.97 × 10-17), the VC (p = 4.404 × 10-178), and both combined (p = 7.74 × 10-199). CONCLUSIONS: RelCC of leukocyte subsets in PB of HNSCC-CPs differ significantly from those of HAs. A score based on MGR, clMMR, MLR, and MTLR allows for accurate discrimination.

Engineered cyclodextrin glucanotransferases from Bacillus sp. G-825-6 produce large-ring cyclodextrins with high specificity.

Cyclodextrin glucanotransferases (CGTases) synthesize cyclic oligosaccharides (cyclodextrins, CD) from starch. A CGTase from Bacillus sp. G-825-6 was engineered by site-directed mutagenesis at two positions by the construction of the variants Y183W, Y183R, D358R, Y183W/D358R, and Y183R/D358R. Among CD composed of 7-12 glucose units (CD7-CD12), Y183W mainly produced CD8. Y183R had completely lost its ability to synthesize CD7, and CD8 and the larger CD were the only cyclic oligosaccharides produced. D358R also formed mainly CD8-CD12 during a reaction time of 24 hr. The double mutant Y183W/D358R showed combined characteristics of the single mutations with very low CD7 cyclization activity and an increased formation of the larger CD. The results show that CGTases synthesizing mainly CD8-CD12 can be constructed allowing a convenient production of larger CD in significant amounts as host molecules in supramolecular complexing reactions.

Two-Color Analysis of Leukocytes Labeled by Modified RBCs and Their Fragments.

Red blood cells (RBCs) are attractive tools for surface modification to adhere specifically to molecules, cellular fragments (e.g., microvesicles), or whole cells for potential use in bioanalytical assays or as a delivery vehicle in targeted therapy. Within this study, we have loaded RBCs with fluorochrome-conjugated antibodies (Ab) against CD45 and CD22 leukocyte markers and evaluated the conjugation process by microscopy. We have assessed the potential application of RBCs fragments generated from conjugated RBCs for targeting Cyto-Trol control cells by flow cytometric (FCM) approaches. Based on their scattering and fluorescence characteristics (FITC and PE expression), modified RBCs and their fragments, Cyto-Trol cells, and clusters of both were distinguished by two color FCM analysis. Fragments with anti-human Kallestad Ab as a nonspecific FITC conjugate had less than 20% binding to Cyto-Trol controls compared to CD45-FITC Ab conjugate with nearly 100% binding capacity. Cyto-Trol-microvesicle-clusters were more than 45% positive for either FITC or PE. Anti-CD22-PE modified RBCs fragments were also useful in staining and showing about 19.5% positively stained events in the Cyto-Trol region. The proof-of-concept shows, that specific antibody can be attached to RBCs, and generated fragments can be useful to stain target cells for FCM analysis. © 2018 International Society for Advancement of Cytometry.

Fast RBC loading by fluorescent antibodies and nuclei staining dye and their potential bioanalytical applications.

This study was designed to load different antibodies (Abs) and a fluorescent dye onto the red blood cell (RBC) surface. We have used fluorescein isothiocyanate (FITC)-conjugate anti-human Ab, CD22-PE (B-cell marker-phycoerythrin Ab), and 4',6-diamidino-2-phenylindole (DAPI) for insertion over the RBC surface. In a first step, conjugation experiments were performed: in dimethyl sulfoxide (DMSO), RBCs were conserved and modified by succinic anhydride to create an additional -COOH group, and then activated with 3-(3-dimethylaminopropyl)carbodiimide-N-hydroxysuccinimide (EDC-NHS) in 2-(N-morpholino) ethanesulfonic acid hydrate buffer for insertion of labeled Abs or DAPI. In a second step, fluorescence signals were evaluated by microscopy and the mean fluorescence intensities of cell lysates were measured by spectrofluorometry. The results showed clear evidence for adsorption of FITC- and PE-labeled Abs to activated conserved RBCs. DAPI was adsorbed well also to DMSO-conserved RBCs without the need for an activation step. The DMSO conservation step was enough to create reactive RBCs for insertion of specific Abs and fluorescent dyes. The additional modification by succinic anhydride and activation with EDC-NHS resulted in two- to seven-fold increase in fluorescence signals, indicating a much higher RBC loading capacity. These Ab- and fluorescent dye-functionalized RBCs have potentially high application in developing new biomedical diagnostic and in vitro assay techniques.

Three-dimensional imaging technologies: a priority for the advancement of tissue engineering and a challenge for the imaging community.

Tissue engineering/regenerative medicine (TERM) is an interdisciplinary field that applies the principle of engineering and life sciences to restore/replace damaged tissues/organs with in vitro artificially-created ones. Research on TERM quickly moves forward. Today newest technologies and discoveries, such as 3D-/bio-printing, allow in vitro fabrication of ex-novo made tissues/organs, opening the door to wide and probably never-ending application possibilities, from organ transplant to drug discovery, high content screening and replacement of laboratory animals. Imaging techniques are fundamental tools for the characterization of tissue engineering (TE) products at any stage, from biomaterial/scaffold to construct/organ analysis. Indeed, tissue engineers need versatile imaging methods capable of monitoring not only morphological but also functional and molecular features, allowing three-dimensional (3D) and time-lapse in vivo analysis, in a non-destructive, quantitative, multidimensional analysis of TE constructs, to analyze their pre-implantation quality assessment and their fate after implantation. This review focuses on the newest developments in imaging technologies and applications in the context of requirements of the different steps of the TERM field, describing strengths and weaknesses of the current imaging approaches.

Trypan blue as an affordable marker for automated live-dead cell analysis in image cytometry.

The aim of the present study was to combine image cytometry and trypan blue (TB) exclusion staining for a reproducible high-throughput detection of dead cells, enabling TB as an inexpensive marker, to be affordable for many studies and creating the possibility to combine fluorochromes without or with less spectral overlap. Capillary blood was drawn from a healthy volunteer, red blood cells were lysed and leukocyte cell death was induced. Samples were stained with CD45-FITC, CD14-PE, TB and DAPI, and then analyzed using image cytometry (iCys). TB quenching control tests were performed using DAPI and CD45-FITC. Images were generated in .TIF and .JPEG format using iCys image cytometer. The images were analyzed using CellProfiler (CP) modules to optimize the analysis based on the aims of each phase of this study. CellProfiler Analyst (CPA) was used to classify cells throughout machine learning and to calculate sensibility of the classification. A sensitivity of 0.94 for dead cells and 0.99 for live cells was calculated using CPA. We did not see any quenching effects of the FITC staining. DAPI signal was reduced in the presence of TB. The results of the present study revealed that TB serves as a dead cell marker in an image cytometric analysis, being able to be combined with other fluorescence markers without loss of fluorescence intensity signal or overlapping emission spectrum. SCANNING 38:857-863, 2016. © 2016 Wiley Periodicals, Inc.

Dendritic Cells in the Context of Human Tumors: Biology and Experimental Tools.

Dendritic cells (DC) are the most potent and versatile antigen-presenting cells (APC) in the immune system. DC have an exceptional ability to comprehend the immune context of a captured antigen based on molecular signals identified from its vicinity. The analyzed information is then conveyed to other immune effector cells. Such capability enables DC to play a pivotal role in mediating either an immunogenic response or immune tolerance towards an acquired antigen. This review summarizes current knowledge on DC in the context of human tumors. It covers the basics of human DC biology, elaborating on the different markers, morphology and function of the different subsets of human DC. Human blood-borne DC are comprised of at least three subsets consisting of one plasmacytoid DC (pDC) and two to three myeloid DC (mDC) subsets. Some tissues have unique DC. Each subset has a different phenotype and function and may induce pro-tumoral or anti-tumoral effects. The review also discusses two methods fundamental to the research of DC on the single-cell level: multicolor flow cytometry (FCM) and image-based cytometry (IC). These methods, along with new genomics and proteomics tools, can provide high-resolution information on specific DC subsets and on immune and tumor cells with which they interact. The different layers of collected biological data may then be integrated using Immune-Cytomics modeling approaches. Such novel integrated approaches may help unravel the complex network of cellular interactions that DC carry out within tumors, and may help harness this complex immunological information into the development of more effective treatments for cancer.

Inflammation in tissue engineering: The Janus between engraftment and rejection.

Tissue engineering (TE) for tissue and organ regeneration or replacement is generally performed with scaffold implants, which provide structural and molecular support to in vitro seeded or in vivo recruited cells. TE implants elicit the host immune response, often resulting in engraftment impediment or rejection. Besides this negative effect, however, the immune system components also yield a positive influence on stem cell recruitment and differentiation, allowing tissue regeneration and healing. Thus, a balanced cooperation between proinflammatory and proresolution players of the immune response is an essential element of implant success. In this context, macrophage plasticity plays a fundamental role. Therefore modulating the immune response, instead of immune suppressing the host, might be the best way to successfully implant TE tissues or organs. In particular, it is becoming evident that the scaffold, immune, and stem cells are linked by a three-way interaction, and many efforts are being made for scaffold-appropriate design and functionalization in order to drive the inflammation process toward regeneration, vascularization, and implant success. This review discusses current and potential strategies for inflammation modulation to aid engraftment and regeneration, supporting the concept that quality, and not quantity, of inflammation might influence implant success.

Detection of gold nanorods uptake by macrophages using scattering analyses combined with diffusion reflection measurements as a potential tool for in vivo atherosclerosis tracking.

In this study, we report a potential noninvasive technique for the detection of vulnerable plaques using scatter analyses with flow cytometry (FCM) method combined with the diffusion reflection (DR) method. The atherosclerotic plaques are commonly divided into two major categories: stable and vulnerable. The vulnerable plaques are rich with inflammatory cells, mostly macrophages (MΦ), which release enzymes that break down collagen in the cap. The detection method is based on uptake of gold nanorods (GNR) by MΦ. The GNR have unique optical properties that enable their detection using the FCM method, based on their scattering properties, and using the DR method, based on their unique absorption properties. This work demonstrates that after GNR labeling of MΦ, 1) the FCM scatter values increased up to 3.7-fold with arbitrary intensity values increasing from 1,110 to 4,100 and 2) the DR slope changed from an average slope of 0.196 (MΦ only) to an average slope of 0.827 (MΦ labeled with GNR) (P<0.001 for both cases). The combination of FCM and DR measurements provides a potential novel, highly sensitive, and noninvasive method for the identification of atherosclerotic vulnerable plaques, aimed to develop a potential tool for in vivo tracking.

The LIFE-Adult-Study: objectives and design of a population-based cohort study with 10,000 deeply phenotyped adults in Germany.

BACKGROUND: The LIFE-Adult-Study is a population-based cohort study, which has recently completed the baseline examination of 10,000 randomly selected participants from Leipzig, a major city with 550,000 inhabitants in the east of Germany. It is the first study of this kind and size in an urban population in the eastern part of Germany. The study is conducted by the Leipzig Research Centre for Civilization Diseases (LIFE). Our objective is to investigate prevalences, early onset markers, genetic predispositions, and the role of lifestyle factors of major civilization diseases, with primary focus on metabolic and vascular diseases, heart function, cognitive impairment, brain function, depression, sleep disorders and vigilance dysregulation, retinal and optic nerve degeneration, and allergies. METHODS/DESIGN: The study covers a main age range from 40-79 years with particular deep phenotyping in elderly participants above the age of 60. The baseline examination was conducted from August 2011 to November 2014. All participants underwent an extensive core assessment programme (5-6 h) including structured interviews, questionnaires, physical examinations, and biospecimen collection. Participants over 60 underwent two additional assessment programmes (3-4 h each) on two separate visits including deeper cognitive testing, brain magnetic resonance imaging, diagnostic interviews for depression, and electroencephalography. DISCUSSION: The participation rate was 33 %. The assessment programme was accepted well and completely passed by almost all participants. Biomarker analyses have already been performed in all participants. Genotype, transcriptome and metabolome analyses have been conducted in subgroups. The first follow-up examination will commence in 2016.

Stepwise error-prone PCR and DNA shuffling changed the pH activity range and product specificity of the cyclodextrin glucanotransferase from an alkaliphilic Bacillus sp.

Cyclodextrin glucanotransferase (EC 2.4.1.19) from the alkaliphilic Bacillus sp. G-825-6 converts starch mainly to γ-cyclodextrin (CD8). A combination of error-prone PCR and DNA shuffling was used to obtain variants of this enzyme with higher product specificity for CD8 and a broad pH activity range. The variant S54 with seven amino acid substitutions showed a 1.2-fold increase in CD8-synthesizing activity and the product ratio of CD7:CD8 was shifted to 1:7 compared to 1:3 of the wild-type enzyme. Nine amino acid substitutions of the cyclodextrin glucanotransferase were performed to generate the variant S35 active in a pH range 4.0-10.0. Compared to the wild-type enzyme which is inactive below pH 6.0, S35 retained 70% of its CD8-synthesizing activity at pH 4.0.

Nanoparticle uptake by macrophages in vulnerable plaques for atherosclerosis diagnosis.

The composition of atherosclerotic (AS) plaques is crucial concerning rupture, thrombosis and clinical events. Two plaque types are distinguished: stable and vulnerable plaques. Vulnerable plaques are rich in inflammatory cells, mostly only M1 macrophages, and are highly susceptible to rupture. These plaques represent a high risk particularly with the standard invasive diagnosis by coronary angiography. So far there are no non-invasive low-risk clinical approaches available to detect and distinguish AS plaque types in vivo. The perspective review introduces a whole work-flow for a novel approach for non-invasive detection and classification of AS plaques using the diffusion reflection method with gold nanoparticle loaded macrophages in combination with flow and image cytometric analysis for quality assurance. Classical biophotonic methods for AS diagnosis are summarized. Phenotyping of monocytes and macrophages are discussed for specific subset labelling by nanomaterials, as well as existing studies and first experimental proofs of concept for the novel approach are shown. In vitro and in vivo detection of NP loaded macrophages (MΦ). Different ways of MΦ labelling include (1) in vitro labelling in suspension (whole blood or buffy coat) or (2) labelling of short-term MΦ cultures with re-injection of MΦ-NP into the animal to detect migration of the cells in the plaques and (3) in vivo injection of NP into the organism.

Reference intervals for leukocyte subsets in adults: Results from a population-based study using 10-color flow cytometry.

BACKGROUND: Reference intervals for leukocyte subsets from peripheral blood are helpful for the understanding of disease states and therapy effects. METHODS: We performed in-depth immunophenotyping for 608 healthy German adults from the Leipzig region from 40 to 79 years by 10-color flow cytometry (FCM) to gain reference information for various leukocyte subsets including subsets of granulocytes, monocytes and lymphocytes. RESULTS: First, we derived gender- and age-specific reference intervals for males and females from 40 to 59 and from 60 to 79 years, respectively. Second, we further investigated the influence of gender and age on leukocyte counts. We found significantly higher cell counts for monocytes (P < 0.001) and NK cells (P < 0.001) in men, whereas women had higher counts for B cells (P < 0.001), Th cells (P < 0.001) and regulatory T cells (P = 0.008). Furthermore, with increasing age, a decrease in Tc cells (about 8% within 5 years) and an increase in NK cells (<4% within 5 years) were observed. CONCLUSION: In future research, it should be investigated whether these are real ageing effects that can be confirmed in longitudinal studies. Furthermore, it is important to understand if the Tc cell count drop is functionally compensated by the increase of NK cells.

Cytometric patterns reveal growth states of Shewanella putrefaciens.

Bacterial growth is often difficult to estimate beyond classical cultivation approaches. Low cell numbers, particles or coloured and dense media may disturb reliable growth assessment. Further difficulties appear when cells are attached to surfaces and detachment is incomplete. Therefore, flow cytometry was tested and used for analysis of bacterial growth on the single-cell level. Shewanella putrefaciens was cultivated as a model organism in planktonic or biofilm culture. Materials of smooth and rough surfaces were used for biofilm cultivation. Both aerobic and anaerobic as well as feast and famine conditions were applied. Visualization of growth was also done using Environmental Scanning and Phase Contrast Microscopy. Bioinformatic tools were applied for data interpretation. Cytometric proliferation patterns based on distributions of DNA contents per cell corresponded distinctly to the various lifestyles, electron acceptors and substrates tested. Therefore, cell cycling profiles of S. putrefaciens were found to mirror growth conditions. The cytometric patterns were consistently detectable with exception of some biofilm types whose resolution remained challenging. Corresponding heat maps proved to be useful for clear visualization of growth behaviour under all tested conditions. Therefore, flow cytometry in combination with bioinformatic tools proved to be powerful means to determine various growth states of S. putrefaciens, even in constrained environments. The approach is universal and will also be applicable for other bacterial species.