scholarly journals Development of an advanced flow cytometry based high-resolution immunophenotyping method to benchmark early immune response in dairy cows

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Sabine Farschtschi ◽  
Martin Mattes ◽  
Alex Hildebrandt ◽  
Dapi Chiang ◽  
Benedikt Kirchner ◽  
...  

AbstractThe determination of the somatic cell count of a milk sample is one of the most common methods to monitor udder health of a dairy cow. However, this procedure does not take into account the fact that cells in milk present a great variety of different cell types. The objective of our study was to establish a high-resolution differential cell count (HRDCC) by means of flow cytometry in blood and milk. We were able to detect ten subpopulations among the three main populations of immune cells and to determine their viability. Additionally, blood samples were analyzed for common laboratory biomarkers, i.e. differential blood counts, haptoglobin levels and several metabolic parameters. In this first feasibility study, we used three different vaccines to stimulate the immune system of five healthy cows each. Samples were collected shortly before, in between and after the vaccinations. Using multivariate statistical methods we saw a diagnostic benefit when HRDCCs were included compared to only the standard laboratory parameters. The impacts of all three vaccinations on the immune system were visible in blood HRDCCs as well as in milk HRDCCs. Cluster of Differentiation 8+ (CD8+) T cells, B cells and monocyte/macrophage subpopulations were among the most important and statistically relevant parameters for all treatments in both biofluids. Moreover, in one of the treatment groups intermediate monocytes showed a significant increase after both vaccinations. Although the use of HRDCC in blood or milk was shown to be highly relevant for early systemic diagnostic, to confirm these subpopulations further investigations in cows of different breed, lactation stage or health status are required.

2008 ◽  
Vol 75 (2) ◽  
pp. 225-232 ◽  
Author(s):  
Cordula Koess ◽  
Joern Hamann

Subclinical mastitis is a costly disease and its diagnosis is difficult. Besides the somatic cell count (SCC) and bacteriology, the differential inflammatory cell count (DICC) is a meaningful tool for mastitis detection. As microscopy is very subjective because of the low number of events to be counted, flow cytometry has often been proposed for the differentiation of milk cells. The objective of this study was to determine whether it is possible to identify subclinical mastitis in cattle at an early stage by a simple and fast flow cytometric method. The aim was to identify the main leucocyte populations in flow cytometric dotplots (polymorphonuclear neutrophils (PMN), lymphocytes and macrophages) and, with these, to elaborate a method of mastitis prognostics. Milk from 15 German Holstein cows was sampled in cross-sectional studies and SCC determined. After preparation, the milk cells were incubated with different specific antibodies that bind to different cell types and also to propidium iodide (PI), which differs between viable and non-viable cells. This procedure made it possible to localize cell types in a flow cytometric dot plot and to differentiate between viable and non-viable PMN. Percentages of viable PMN can be determined by a procedure consisting of a simple centrifugation, incubation with PI, and flow cytometric measurement. So it is possible to quickly determine the stage of the inflammation even in quarters with a low SCC.


2020 ◽  
Vol 15 (3) ◽  
pp. 187-201 ◽  
Author(s):  
Sunil K. Dubey ◽  
Amit Alexander ◽  
Munnangi Sivaram ◽  
Mukta Agrawal ◽  
Gautam Singhvi ◽  
...  

Damaged or disabled tissue is life-threatening due to the lack of proper treatment. Many conventional transplantation methods like autograft, iso-graft and allograft are in existence for ages, but they are not sufficient to treat all types of tissue or organ damages. Stem cells, with their unique capabilities like self-renewal and differentiate into various cell types, can be a potential strategy for tissue regeneration. However, the challenges like reproducibility, uncontrolled propagation and differentiation, isolation of specific kinds of cell and tumorigenic nature made these stem cells away from clinical application. Today, various types of stem cells like embryonic, fetal or gestational tissue, mesenchymal and induced-pluripotent stem cells are under investigation for their clinical application. Tissue engineering helps in configuring the stem cells to develop into a desired viable tissue, to use them clinically as a substitute for the conventional method. The use of stem cell-derived Extracellular Vesicles (EVs) is being studied to replace the stem cells, which decreases the immunological complications associated with the direct administration of stem cells. Tissue engineering also investigates various biomaterials to use clinically, either to replace the bones or as a scaffold to support the growth of stemcells/ tissue. Depending upon the need, there are various biomaterials like bio-ceramics, natural and synthetic biodegradable polymers to support replacement or regeneration of tissue. Like the other fields of science, tissue engineering is also incorporating the nanotechnology to develop nano-scaffolds to provide and support the growth of stem cells with an environment mimicking the Extracellular matrix (ECM) of the desired tissue. Tissue engineering is also used in the modulation of the immune system by using patient-specific Mesenchymal Stem Cells (MSCs) and by modifying the physical features of scaffolds that may provoke the immune system. This review describes the use of various stem cells, biomaterials and the impact of nanotechnology in regenerative medicine.


Biomedicines ◽  
2021 ◽  
Vol 9 (2) ◽  
pp. 124
Author(s):  
Jaco Botha ◽  
Haley R. Pugsley ◽  
Aase Handberg

Flow cytometry remains a commonly used methodology due to its ability to characterise multiple parameters on single particles in a high-throughput manner. In order to address limitations with lacking sensitivity of conventional flow cytometry to characterise extracellular vesicles (EVs), novel, highly sensitive platforms, such as high-resolution and imaging flow cytometers, have been developed. We provided comparative benchmarks of a conventional FACS Aria III, a high-resolution Apogee A60 Micro-PLUS and the ImageStream X Mk II imaging flow cytometry platform. Nanospheres were used to systematically characterise the abilities of each platform to detect and quantify populations with different sizes, refractive indices and fluorescence properties, and the repeatability in concentration determinations was reported for each population. We evaluated the ability of the three platforms to detect different EV phenotypes in blood plasma and the intra-day, inter-day and global variabilities in determining EV concentrations. By applying this or similar methodology to characterise methods, researchers would be able to make informed decisions on choice of platforms and thereby be able to match suitable flow cytometry platforms with projects based on the needs of each individual project. This would greatly contribute to improving the robustness and reproducibility of EV studies.


2000 ◽  
Vol 16 (3-4) ◽  
pp. 131-133 ◽  
Author(s):  
S. Feki ◽  
H. El Omri ◽  
M. A. Laatiri ◽  
S. Ennabli ◽  
K. Boukef ◽  
...  

The precision of immunological characterization of leukemias was improved by a certain number of technical innovations, particularly hybridoma production and standardization, resulting in monoclonal antibodies and definition of recognised cellular antigens (designated by CD: Cluster of Differentiation).The aim of this work was to determine the immunophenotyping profile of patients with leukemia, by means of a flow cytometric method: 66 blood samples coming from leukemic persons in the Sahel region were studied by flow cytometry, using about thirty monoclonal antibodies all marked with a fluorochrome, in one or two colour systems to assess their distribution according to type (lymphoid B or T / myeloid) and age, and to search for possible co-expressions of markers of different lineages.The marked preponderance of childhood B-ALL in our series is, at least partly, attributable to the age distribution of the Tunisian population. In agreement with studies from other countries, the majority of AML cases occurred among adults. A high proportion of AML cases in our series co-expressed markers of other lineages. Overall, accurate classification of acute leukemias was possible from a simple peripheral blood sample in 62 of 66 cases (93.9%).


2017 ◽  
Vol 55 ◽  
pp. S98-S99
Author(s):  
P. Font ◽  
D. Subira ◽  
S. Matarraz ◽  
C. Benavente ◽  
T. Cedena ◽  
...  

2017 ◽  
Author(s):  
◽  
Tuan Anh Trieu

[ACCESS RESTRICTED TO THE UNIVERSITY OF MISSOURI AT AUTHOR'S REQUEST.] Different cell types of an organism have the same DNA sequence, but they can function differently because their difference in 3D organization allows them to express different genes and has different cellular functions. Understanding the 3D organization of the genome is the key to understand functions of the cell. Chromosome conformation capture techniques like Hi-C and TCC that can capture interactions between proximal chromosome fragments have allowed the study of 3D genome organization in high resolution and high through-put. My work focuses on developing computational methods to reconstruct 3D genome structures from Hi-C data. I presented three methods to reconstruct 3D genome and chromosome structures. The first method can build 3D genome models from soft constraints of contacts and non-contacts. This method utilizes the concept of contact and non-contact to reconstruct 3D models without translating interaction frequencies into physical distances. The translation is commonly used by other methods even though it makes a strong assumption about the relationship between interaction frequencies and physical distances. In synthetic dataset, when the relationship was known, my method performed comparably with other methods assuming the relationship. This shows the potential of my method for real Hi-C datasets where the relationship is unknown. The limitation of the method is that it has parameters requiring manual adjustment. I developed the second method to reconstruct 3D genome models. This method utilizes a commonly used function to translate interaction frequencies to physical distances to build 3D models. I proposed a novel way to derive soft constraints to handle inconsistency in the data and to make the method robust. Building 3D models at high resolution is a more challenging problem as the number of constraints is small and the feasible space is larger. I introduced a third method to build 3D chromosome models at high resolution. The method reconstructs models at low resolution and then uses them to guide the reconstruction of models at high resolution. The last part of my work is the development of a comprehensive tool with intuitive graphic user interface to analyze Hi-C data, reconstruct and analyze 3D models.


2021 ◽  
Author(s):  
Romina Mitarotonda ◽  
Martín Saraceno ◽  
Marcos Todone ◽  
Exequiel Giorgi ◽  
Emilio L Malchiodi ◽  
...  

Aim: Nanoparticles (NPs) interaction with immune system is a growing topic of study. Materials & methods: Bare and amine grafted silica NPs effects on monocytes/macrophages cells were analyzed by flow cytometry, MTT test and LIVE/DEAD® viability/cytotoxicity assay. Results: Bare silica NPs inhibited proliferation and induced monocyte/macrophages activation (increasing CD40/CD80 expression besides pro-inflammatory cytokines and nitrite secretion). Furthermore, silica NPs increased cell membrane damage and reduced the number of living cells. In contrast, amine grafted silica NPs did not alter these parameters. Conclusion: Cell activation properties of bare silica NPs could be hindered after grafting with amine moieties. This strategy is useful to tune the immune system stimulation by NPs or to design NPs suitable to transport therapeutic molecules.


Author(s):  
Rosanna Serafini ◽  
Dickson D. Varner ◽  
Charles C. Love

2011 ◽  
Vol 2011 ◽  
pp. 1-7 ◽  
Author(s):  
Jamal Kamalov ◽  
David O. Carpenter ◽  
Irina Birman

The effects of low concentrations of aluminum chloride on thymocytes and lymphocytes acutely dissociated from young mice were studied using flow cytometry with a DNA-binding dye. We demonstrate a rapid and dose-dependent injury in murine thymocytes and lymphocytes resulting from exposure to aluminum, as indicated by an increase in the entry into the cell of the DNA-binding dye, propidium iodine. A 60-minute exposure to 10 μM AlCl3caused damage of about 5% of thymocytes, while 50% were injured after 10 minutes at 20 μM. Nearly all thymocytes showed evidence of damage at 30 μM AlCl3after only 5 minutes of incubation. In lymphocytes, injury was observed at 15 μM AlCl3and less than 50% of cells were injured after a 60-minute exposure to 20 μM. Injury only rarely proceeded to rapid cell death and was associated with cell swelling. These results suggest that aluminum has cytotoxic effects on cells of the immune system.


2018 ◽  
Vol 20 (1) ◽  
pp. 19 ◽  
Author(s):  
Yadong Wei ◽  
Krishan Chhiba ◽  
Fengrui Zhang ◽  
Xujun Ye ◽  
Lihui Wang ◽  
...  

Sialic acid-binding Ig-like lectin 8 (Siglec-8) is expressed on the surface of human eosinophils, mast cells, and basophils—cells that participate in allergic and other diseases. Ligation of Siglec-8 by specific glycan ligands or antibodies triggers eosinophil death and inhibits mast cell degranulation; consequences that could be leveraged as treatment. However, Siglec-8 is not expressed in murine and most other species, thus limiting preclinical studies in vivo. Based on a ROSA26 knock-in vector, a construct was generated that contains the CAG promoter, a LoxP-floxed-Neo-STOP fragment, and full-length Siglec-8 cDNA. Through homologous recombination, this Siglec-8 construct was targeted into the mouse genome of C57BL/6 embryonic stem (ES) cells, and chimeric mice carrying the ROSA26-Siglec-8 gene were generated. After cross-breeding to mast cell-selective Cre-recombinase transgenic lines (CPA3-Cre, and Mcpt5-Cre), the expression of Siglec-8 in different cell types was determined by RT-PCR and flow cytometry. Peritoneal mast cells (dual FcεRI+ and c-Kit+) showed the strongest levels of surface Siglec-8 expression by multicolor flow cytometry compared to expression levels on tissue-derived mast cells. Siglec-8 was seen on a small percentage of peritoneal basophils, but not other leukocytes from CPA3-Siglec-8 mice. Siglec-8 mRNA and surface protein were also detected on bone marrow-derived mast cells. Transgenic expression of Siglec-8 in mice did not affect endogenous numbers of mast cells when quantified from multiple tissues. Thus, we generated two novel mouse strains, in which human Siglec-8 is selectively expressed on mast cells. These mice may enable the study of Siglec-8 biology in mast cells and its therapeutic targeting in vivo.


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