Characterization Of Leukocytes Research Articles

Characterization of Leukocyte- and Platelet-Rich Plasma Derived from Female Collage Athletes: A Cross-Sectional Cohort Study Focusing on Growth Factor, Inflammatory Cytokines, and Anti-Inflammatory Cytokine Levels.

Platelet-rich plasma (PRP) has been increasingly used in sports medicine owing to its various advantages. The purpose of our project was to standardize the parameters before performing large-scale clinical trials in the near future to precisely evaluate individual PRP quality. To examine the effects of regular exercise on PRP quality, this study focused on young female athletes, who have been relatively less studied. Blood samples were obtained from female college athletes (n = 35) and ordinary healthy adults (n = 30), which were considered as controls, and leukocyte-rich PRP (L-PRP) was prepared manually. Body composition indices were determined using a bathroom weight scale equipped with an impedance meter. Growth factors and cytokines were quantified using ELISA kits. Platelet-derived growth factor-BB (PDGF-BB) and Transforming-growth factors β1 (TGFβ1) levels (per platelet) in L-PRP were significantly lower in female athletes than in controls. In contrast, Interleukin-1β and Interleukin 1 receptor antagonist (IL-1RA) levels (per platelet and L-PRP) in L-PRP were significantly higher in athletes, and this difference was more prominent in IL-1RA. These findings suggest that L-PRP from athletes may facilitate the inflammatory phase of the healing process by regulating the pro-inflammatory and anti-inflammatory balance. These chemical compositions can be adopted as "must-check" parameters to characterize individual PRP preparations prior to clinical trials.

Preparation and characterization of leukocyte- and platelet-rich fibrin membrane derived from cats' blood.

Autologous platelet concentrates have been used in regenerative medicine in humans due to the abundance of growth factors, but there are only a few reports in small animals. This study aimed to prepare and characterize a leukocyte and platelet-rich fibrin membrane (L-PRF) produced with blood obtained from cats. Thirteen client-owned healthy adult Maine Coon cats were enrolled. The blood samples were collected and centrifuged at 650g for 12 min using a centrifuge specifically designed for this application. The L-PRF clot was removed from the tube and red blood cell base layer was separated, leaving buffy coat intact. After this, L-PRF clot was compressed by specialized metal plate for 30-60 s, and L-PRF membrane was obtained. Light microscopy examination of the membranes showed three distinct layers: white part, buffy coat, and red part. Immunohistochemical analysis demonstrated expression of vascular endothelial growth factor and platelet derived growth factor. The scanning electron microscopy showed that three-dimensional architecture of fibrin network was more compact in the area near the buffy coat. In conclusion, the method used allowed the characterization of the L-PRF membrane composition, which presented cell types and fibrin network architecture similar to those described in the human species.

Characterization of Leukocytes From HIV-ART Patients Using Combined Cytometric Profiles of 72 Cell Markers.

Motivation: Mass cytometry is a technique used to measure the intensity levels of proteins expressed by cells, at a single cell resolution. This technique is essential to characterize the phenotypes and functions of immune cell populations, but is currently limited to the measurement of 40 cell markers that restricts the characterization of complex diseases. However, algorithms and multi-tube cytometry techniques have been designed for combining phenotypic information obtained from different cytometric panels. The characterization of chronic HIV infection represents a good study case for multi-tube mass cytometry as this disease triggers a complex interactions network of more than 70 cell markers.Method: We collected whole blood from non-viremic HIV-infected patients on combined antiretroviral therapies and healthy donors. Leukocytes from each individual were stained using three different mass cytometry panels, which consisted of 35, 32, and 33 cell markers. For each patient and using the CytoBackBone algorithm, we combined phenotypic information from three different antibody panels into a single cytometric profile, reaching a phenotypic resolution of 72 markers. These high-resolution cytometric profiles were analyzed using SPADE and viSNE algorithms to decipher the immune response to HIV.Results: We detected an upregulation of several proteins in HIV-infected patients relative to healthy donors using our profiling of 72 cell markers. Among them, CD11a and CD11b were upregulated in PMNs, monocytes, mDCs, NK cells, and T cells. CD11b was also upregulated on pDCs. Other upregulated proteins included: CD38 on PMNs, monocytes, NK cells, basophils, B cells, and T cells; CD83 on monocytes, mDCs, B cells, and T cells; and TLR2, CD32, and CD64 on PMNs and monocytes. These results were validated using a mass cytometry panel of 25 cells markers.Impacts: We demonstrate here that multi-tube cytometry can be applied to mass cytometry for exploring, at an unprecedented level of details, cell populations impacted by complex diseases. We showed that the monocyte and PMN populations were strongly affected by the HIV infection, as CD11a, CD11b, CD32, CD38, CD64, CD83, CD86, and TLR2 were upregulated in these populations. Overall, these results demonstrate that HIV induced a specific environment that similarly affected multiple immune cells.

Chemokine in der Augenheilkunde

Chemokines are chemotactically active cytokines, which coordinate the distribution of immune cells within the body and also regulate the migration of leukocytes in malignant and inflammatory processes. Chemokines are aheterogeneous group of short-chain proteins that are divided into different subgroups on the basis of their structure. In addition to the chemokines (ligands) various chemokine receptors also exist. The chemokine system is given its complexity by the high redundancy of ligand-receptor interactions: one single ligand can bind to different receptors and asingle receptor can interact with different ligands. In terms of receptors, distinct immune cell types have characteristic receptor expression patterns, which can be used for the immunological characterization of leukocytes. Important basic research is currently leading to abetter understanding of the chemokine system. The essential importance of the chemokine system in various diseases of the anterior and posterior eye segments is becoming increasingly apparent. The following synopsis explains the individual clinical aspects as well as the underlying scientific work in the context of "chemokines in ophthalmology".

Isolation and characterization of leukocytes from the placental bed following normal term Cesarean section delivery

Isolation and characterization of leukocytes from the placental bed following normal term Cesarean section delivery

Graphene Oxide Nanosheets Modified with Single-Domain Antibodies for Rapid and Efficient Capture of Cells.

Peripheral blood can provide valuable information on an individual's immune status. Cell-based assays typically target leukocytes and their products. Characterization of leukocytes from whole blood requires their separation from the far more numerous red blood cells.1 Current methods to classify leukocytes, such as recovery on antibody-coated beads or fluorescence-activated cell sorting require long sample preparation times and relatively large sample volumes.2 A simple method that enables the characterization of cells from a small peripheral whole blood sample could overcome limitations of current analytical techniques. We describe the development of a simple graphene oxide surface coated with single-domain antibody fragments. This format allows quick and efficient capture of distinct WBC subpopulations from small samples (∼30 μL) of whole blood in a geometry that does not require any specialized equipment such as cell sorters or microfluidic devices.

Lipocalin-2 (LCN2) in experimental liver injury and human liver diseases

Background: LCN2 is a member of the lipocalin family that is expressed under pernicious conditions such as intoxication, infection, inflammation and cellular stress. Sustained LCN2 expression is induced in experimental liver injury. Immunohistochemistry and primary liver cell isolation identified injured hepatocytes as the main source of LCN2. Methods: LCN2-/- mice were used in experimental models (CCl4, BDL, LPS and ConA) for both acute and chronic liver injury. Liver function analysis, FACS-based liver leukocyte characterization, qRT-PCR for chemokine and cytokine expression and Western Blot as well as histology for fibrotic markers was performed. In addition, serum LCN2 from 91 control and 194 liver diseased patients was analyzed in ELISA. Results: Acute single dose CCl4 intoxication showed lipid droplet accumulation in hepatocytes in LCN2-/- mice. Liver leukocyte isolation showed CD45+ and CD11b+F4–80+ to significantly decrease in LCN2-/- mice, while we found no difference after 5 day BDL or during chronic CCl4 application or BDL for 4 weeks. In ConA models however, LCN2-/- mice showed slightly more liver damage and increased values of CD11b+F4–80+, CD45+ and Tregs than wild type animals. Although serum LCN2 levels in diseased humans were higher than in control patients, we found no difference between cirrhotic and non-cirrhotic patients. Patients with Child A and Child C showed higher LCN2 levels compared to control patients that were independent from disease etiology and serum AST and ALT. By contrast, serum LCN2 showed significant correlation with glomerular filtration rate. Conclusion: LCN2 is a fatty acid transport protein that mobilizes fatty acids from hepatocytes. LCN2-/- mice are unable to appropriately respond to acute CCl4 intoxication. LCN2 expression during inflammatory responses in liver depends on the chosen model. It acts as an acute-phase response protein but the expression during chronic liver insult does not correlate to AST and ALT.

Characterization of Leukocyte Mono-immunoglobulin-like Receptor 7 (LMIR7)/CLM-3 as an Activating Receptor: ITS SIMILARITIES TO AND DIFFERENCES FROM LMIR4/CLM-5

Here we characterize leukocyte mono-Ig-like receptor 7 (LMIR7)/CLM-3 and compare it with an activating receptor, LMIR4/CLM-5, that is a counterpart of an inhibitory receptor LMIR3/CLM-1. LMIR7 shares high homology with LMIR4 in the amino acid sequences of its Ig-like and transmembrane domains. Flow cytometric analysis demonstrated that LMIR4 was predominantly expressed in neutrophils, whereas LMIR7 was highly expressed in mast cells and monocytes/macrophages. Importantly, LMIR7 engagement induced cytokine production in bone marrow-derived mast cells (BMMCs). Although FcRγ deficiency did not affect surface expression levels of LMIR7, it abolished LMIR7-mediated activation of BMMCs. Consistently we found significant interaction of LMIR7-FcRγ, albeit with lower affinity compared with that of LMIR4-FcRγ. Our results showed that LMIR7 transmits an activating signal through interaction with FcRγ. In addition, like LMIR4, LMIR7 synergizes with TLR4 in signaling. Analysis of several chimera receptors composed of LMIR4 and LMIR7 revealed these findings: 1) the transmembrane of LMIR7 with no charged residues maintained its surface expression at high levels in the absence of FcRγ; 2) the extracellular juxtamembrane region of LMIR7 had a negative effect on its surface expression levels; and 3) the strong interaction of LMIR4 with FcRγ depended on the extracellular juxtamembrane region as well as the transmembrane domain of LMIR4. Thus, LMIR7 shares similarities with LMIR4, although they are differentially regulated in their distribution, expression, and function.

The early inflammatory response to bites of Leishmania major infected Phlebotomus duboscqi sand flies in naïve and pre-exposed mice. (94.15)

Abstract Pre-exposure to uninfected sand fly bites (USFB) protects against Leishmania major infection. Little is known about the initial immune response responsible for this protection. During blood feeding, the insect sand fly inoculates Leishmania parasites into the skin alongside saliva. Here, we explore the early inflammatory response in the skin of pre-exposed (PE) mice challenged with L. major-infected P. duboscqi flies. The relative expression of cytokines and chemokines by skin cells were investigated 2- 48h post-infection using a macroarray (Oligo GEArray®). There is a rapid and strong early expression of inflammatory genes in PE compared to naive mice following challenge. Two to twelve hours post-infection, naïve mice showed an increased expression of MCP-1, MCP-3 and eotaxin that attract macrophages and granulocytes while PE mice upregulated TCA-3, BRAK and I-TAC related to migration of NK cells, macrophages, granulocytes and activated T cells. Characterization of leukocytes by FACS 24-48h post-infection showed a significantly increased recruitment of neutrophils, NK cells, macrophages and CD4+ cells in PE mice. PE mice also presented a higher frequency of IFN-γ producing CD4+ and NK cells 24h post-infection. This suggests that induced cytokines and chemokines are altered by pre-exposure to USFB. Thus, protection from Leishmania infection observed in PE mice may result from Th1 priming by an anti-saliva immunity that acts in concert with a modulated innate immune response.

Lensfree holographic imaging of antibody microarrays for high-throughput detection of leukocyte numbers and function.

Characterization of leukocytes is an integral part of blood analysis and blood-based diagnostics. In the present paper, we combine lensless holographic imaging with antibody microarrays for rapid and multiparametric analysis of leukocytes from human blood. Monoclonal antibodies (Abs) specific for leukocyte surface antigens (CD4 and CD8) and cytokines (TNF-alpha, IFN-gamma, IL-2) were printed in an array so as to juxtapose cell capture and cytokine detection antibody (Ab) spots. Integration of Ab microarrays into a microfluidic flow chamber (4 muL volume) followed by incubation with human blood resulted in capture of CD4 and CD8 T-cells on specific Ab spots. On-chip mitogenic activation of these cells induced release of cytokine molecules that were subsequently captured on neighboring anticytokine Ab spots. The binding of IL-2, TNF-alpha, and IFN-gamma molecules on their respective Ab spots was detected using horseradish peroxidase (HRP)-labeled anticytokine Abs and a visible color reagent. Lensfree holographic imaging was then used to rapidly ( approximately 4 s) enumerate CD4 and CD8 T-lymphocytes captured on Ab spots and to quantify the cytokine signal emanating from IL-2, TNF-alpha, and IFN-gamma spots on the same chip. To demonstrate the utility of our approach for infectious disease monitoring, blood samples of healthy volunteers and human immunodeficiency virus (HIV)-infected patients were analyzed to determine the CD4/CD8 ratio, an important HIV/AIDS diagnostic marker. The ratio obtained by lensfree on-chip imaging of CD4 and CD8 T-cells captured on Ab spots was in close agreement with conventional microscopy-based cell counting. The present paper, describing tandem use of Ab microarrays and lensfree holographic imaging, paves the way for future development of miniature cytometry devices for multiparametric blood analysis at the point of care or in a resource-limited setting.

Analysis of retinal cellular infiltrate in experimental autoimmune uveoretinitis reveals multiple regulatory cell populations

Experimental autoimmune uveoretinitis (EAU) is an animal model for human intraocular inflammatory disease. EAU is induced in B10.RIII mice by immunization with RBP-3 161–180 peptide and intraperitoneal pertussis toxin and is mediated by CD4 + T cells that generate a clinically monophasic disease peaking approximately 2 weeks post-immunization. Collagenase digestion of retinal tissue allowed the quantification and characterization of leukocytes in the inflamed retina during disease progression. Using this method we identified three stages of disease. Initially there is a prodromal phase where we found significant changes in the number of leukocytes in the eye as early as 5 days post-immunization. This effect was, in part, non-antigen specific as a small increase in retinal leukocytes was also observed following immunization with OVA peptide. Following the prodrome there is a primary peak of infiltration including both CD4 + T cells and CD11b + cells. This coincides with an early influx of neutrophils and is associated with a peak in IL-17-producing T cells. The neutrophils in the eye are CD11b + and Gr1 + but can be distinguished from other myeloid cells by their high expression of Ly6G. The remaining CD11b +Gr1 + cells can suppress proliferation and are analogous to myeloid derived suppressor cells which are found in tumors. The inflamed eye also contains a considerable proportion of FoxP3 + regulatory cells. Following peak disease, the retina does not return to its pre-disease phenotype. Instead, fluctuations in infiltrating leukocyte numbers and changes to their relative composition continue, indicating that clinical recovery does not equate to the restoration of a normal retinal leukocyte population.

Garbage in-garbage out: influence of preanalytical variables on hematological analysis

Preanalytical factors have a significant impact on the quality of hematological analyses and must not be disregarded during clinical interpretation of laboratory data. Besides unchangeable physiological variables such as age, sex, race, patient preparation, an expert phlebotomy, rapid transport to the laboratory as well as many other variable factors play a crucial role in obtaining correct laboratory results. More advanced tests (erythrocyte enzymes, evaluation of hemoglobinopathies, molecular biology, flowcytometric characterization of leucocytes etc.) performed by specialized centers require special preanalytical measures. The centers should be consulted about the preanalytical conditions before blood sampling is performed or--if possible--the patient should be sent to these centers for phlebotomy.

A method for the dispersal and characterization of leukocytes from the human female reproductive tract.

The isolation of human female reproductive tract (RT) cells that maintain viability and are representative of the entire population is essential for a thorough evaluation of mucosal immunity in the reproductive tract mucosa. Here, we describe the isolation of RT cells in high yields and with high viability from the Fallopian tube, uterine endometrium, endocervix, ectocervix and vagina. This cell dispersion method uses an enzyme cocktail composed of pancreatin, hyaluronidase, and collagenase (PHC), and employs a 250-microm mesh screen to facilitate cell dispersion. The yields of cells isolated per gram of tissue in the presence of this PHC cocktail were compared and found to be strikingly higher relative to the yields obtained with other enzyme cocktails or in the absence of enzymes. Flow cytometry was used to characterize leukocyte subsets isolated from uterine endometrium in the presence of the various enzyme cocktails. The common leukocyte antigen marker CD45, pan T-cell marker CD3, monocyte/macrophage marker CD14 and B-cell marker CD19 were retained after exposure to the PHC cocktail of enzymes. The expression of CD8 and CD4 was lost after exposure to added enzymes but regained after culture overnight. These studies demonstrate the feasibility of using enzymatic digestion for the isolation of whole populations of Fallopian tube, endometrial, cervical and vaginal cells, including leukocyte subsets in high yields, and provide a foundation for investigating mucosal immune cell function in the human female RT.

Detection of leukocytes in contact with the vessel wall from in vivo microscope recordings using a neural network.

Leukocytes play an important role in the host defense as they may travel from the blood stream into the tissue in reacting to inflammatory stimuli. The leukocyte-vessel wall interactions are studied in post capillary vessels by intravital video microscopy during in vivo animal experiments. Sequences of video images are obtained and digitized with a frame grabber. A method for automatic detection and characterization of leukocytes in the video images is developed. Individual leukocytes are detected using a neural network that is trained with synthetic leukocyte images generated using a novel stochastic model. This model makes it feasible to generate images of leukocytes with different shapes and sizes under various lighting conditions. Experiments indicate that neural networks trained with the synthetic leukocyte images perform better than networks trained with images of manually detected leukocytes. The best performing neural network trained with synthetic leukocyte images resulted in an 18% larger area under the ROC curve than the best performing neural network trained with manually detected leukocytes.

Characterization of leukocytes by filtration of diluted blood.

From the known numbers and properties of normal blood cells, it is apparent that the analysis of the flow of diluted blood, through micropore filters, should allow calculation of the properties of leukocytes without the need for their prior purification. The number and transit time of a "slow" leukocyte population can be deduced by fitting the flow, of diluted blood through 5 microns membranes over about 150 s, to the appropriate mathematical model, which is chosen by the use of a suitable statistical test--the runs test. This population of leukocytes equates numerically with the monocyte population of normal blood; cells have a transit time, through 5 microns pores, of 27.7 +/- 10.9 s. The remaining "fast" leukocytes represent the sum of granulocytes and lymphocytes; their flow properties can be deduced from the measured initial flow rate of diluted blood and the estimated properties of the red blood cells. The properties of the red cells can be assessed from filtration of purified suspensions with any concentration of cells from 0.52 x 10(9)/ml to 3.0 x 10(9)/ml. The transit times for red cells and granulocytes/lymphocytes, in blood diluted with about an equal volume of buffer, are 1.36 +/- 0.17 ms and 1.48 +/- 0.33 s, respectively. The transit times of blood cells, through 5 microns pores, are therefore inversely related to their numbers in blood.

Characterization of leukocytes by filtration of diluted blood

Characterization of leukocytes by filtration of diluted blood

Bovine gut-associated lymphoid tissue — Morphologic and functional studies: I. Isolation and characterization of leukocytes from the epithelium and lamina propria of bovine small intestine

A successful technique for the isolation of highly pure suspensions of viable leukocytes from the small intestine of cattle is described. Procedures ranging from mechanical mincing to enzymatic digestion of tissues were compared. The most reliable and reproducible procedure was the sequential treatment of tissues with dithiothreitol (DTT), ethylenediaminetetraacetic acid (EDTA) in calcium-magnesium-free salt solutions, and collagenase. Two populations of mucosal leukocytes were obtained from the small intestine. One population was derived from within the epithelium (intraepithelial leukocytes, IEL), the second from within the lamina propria (lamina propria leukocytes, LPL). At least 2 × 10 6 viable leukocytes were obtainable from each square centimeter of the intestinal mucosa from either the epithelium or lamina propria. Erythrocyte rosetting and immunofluorescence characterization with conventional antisera and monoclonal antibodies (MAB) demonstrated that IEL were predominantly T cells (60%), with relatively few B cells present (10%), while LPL contained relatively high numbers of B cells (28%) and a reasonable percentage of T cells (45%). Both cell populations proliferate in resposne to stimulation with T and B cell mitogens. Addition of the thiol compound, 2-mercaptoethanol (2-ME) strongly augmented the mitogenic response of both cell isolates. Human recombinant interleukin-2 (hr-IL-2) in the presence or absence of additional stimuli was found to be able to induce the proliferation of both cell types. These results demonstrate that functinal leukocytes can be isolated from the small intestine of cattle, and that they can maintain their responsiveness to both T and B cell mitogens and to exogenous cloned IL-2.

A light and electron microscope study on the phagocytosis of leucocytes in rockfish and rainbow trout.

Identification and characterization of leucocytes in two teleosts, rockfish Sebastes schlegeli and rainbow trout Salmo gairdneri were performed based on their morphology and phagocytic ability with bacteria, Escherichia coli and Staphylococcus aureus and zymosan particles. Cytochemical test for peroxidase was also applied to the leucocytes. Light and electron microscopy revealed that both neutrophils and monocytes in the two teleosts actively phagocytized bacteria. Thrombocytes ingested bacteria, but their phagocytic activity was very low. The process of phagocytosis was observed with neutrophils and monocytes. Fusion of cyto-plasmic granules into phagosomes was observed. Large phagosomes were present in neutrophils but entirely (rockfish) or almost absent (rainbow trout) in monocytes. Cytoplasmic granules in neutrophils of both fishes were positive for peroxidase. The peroxidase reaction product was also observed in phagosomes. Therefore, the process of phagocytosis was considered to be similar to that known with mammalian leucocytes. The fate of bacteria phagocytized by fish leucocytes differed to that in mammals. At even 12 h (rockfish) or 6 h (rainbow trout) after incubation, bacteria in phagosomes were almost intact and they seemed to be digested slowly compared with those ingested in mammalian leucocytes.

Leukocyte Function and Characterization of Leukocyte Glucose-6-phosphate Dehydrogenase in Sicilian Mutants

Extract: Nine Sicilian children known to be deficient in glucose-6-phosphate dehydrogenase (G6PD) were studied to see if there were anomalies of bactericidal activity in peripheral blood phagocytes. The type of deficiency was established. The G6PD levels in the leukocyte were found to be 26% of the controls (0.094 ± 0.03, normal controls 0.360 ± 0.12). The Michaelis constant for NADP and glucose-6-phosphate (G6P) was lower than the control. Conversely, the utilization of the analogous 2-deoxyglucose-6-phosphate (2dG6P) and galactose-6-phosphate (Ga16P) was higher. The thermostability of the enzyme in the deficient subjects was lower and the pH optima (8 and 9.5) were different from the controls. An identical electrophoretic pattern was found in both normal and deficient subjects. The bactericidal activity in the deficient subjects was normal. There was no difference in the results of nitroblue tetrazolium (NBT) tests in either group.Speculation: Although leukocyte G6PD was only one-quarter of the normal level, the phagocytic activity and the NBT test were normal in all subjects studied. It is not clear how such low levels of enzyme allow normal function. Perhaps further investigation under simulated intracellular conditions could give more reliable information about the enzyme activity.

Leukocyte function and characterization of leukocyte glucose-6-phosphate dehydrogenase in Sicilian mutants.

Nine Sicilian children known to be deficient in glucose-6-phosphate dehyrdrogenase (G6PD) were studied to see if there were anomalies of bactericidal activity in peripheral blood phagocytes. The type of deficiency was established. The G6PD levels in the leukocyte were found to be 26% of the controls (0.094 +/- 0.03, normal controls 0.360 +/- 0.12). The Michaelis constant for NADP and glucose-6-phosphate (G6P) was lower than the control. Conversely, the utilization of the analogous 2-deoxyglucose-6-phosphate (2dG6P) and galactose-6-phosphate (Ga16P) was higher. The thermostability of the enzyme in the deficient subjects was lower and the pH optima (8 and 9.5) were different from the controls. An identical electrophoretic pattern was found in both normal and deficient subjects. The bactericidal activity in the deficient subjects was normal. There was no difference in the results of nitroblue tetrazolium (NBT) tests in either group.