Leukocyte Fraction Of Blood Research Articles

DNA methylation profile and expression of the phyllagrin gene in patients with atopic dermatitis: case-control study

Background. Atopic dermatitis (AD) is one of the most common inflammatory skin diseases, affecting up to 20.0% of children and 2.0–8.0% of adults worldwide. Patients with atopic dermatitis usually have dry skin and itching, they are at higher risk of developing asthma, as well as allergic rhinitis. Features of the clinical course of AD are associated with both interaction of genes and influence of environmental factors. Studying epigenetic mechanisms could provide understanding of the most likely mechanisms by which environmental factors influence gene expression and contribute to the development of AD. The aim of the study is to conduct a comprehensive molecular genetic analysis (assessment of the level of global DNA methylation and expression of the filaggrin gene (FLG) in patients with moderate to severe AD. Material and methods: The case-control study was conducted from January 2022, through June 2022. A total of 32 patients with AD and 6 healthy volunteers were recruited. The level of global DNA methylation in the blood of patients with AD and healthy individuals was assessed. The FLG expression was measured by polymerase chain reaction in the leukocyte fraction of blood. A three-stage cycle is used during PCR. To determine the expression level of the FLG gene, total RNA was isolated, followed by a reverse transcription, amplification, and detection reaction. Results. A hypermethylation of the genome in patients with AD is 2.3 times higher than the methylation of genome in controls (p = 0.003),it was also found that the expression level of the FLG gene (exon 3, position 7–8, and exon 1) in the leukocyte fraction of blood in patients with AD did not significantly differ from the control group (p = 0.41). Conclusion. The current study found that the epigenome of AD patients differs from healthy individuals. The study of pathogenetic mechanisms is the basis for the development of new methods of targeted therapy for this socially significant dermatosis.

Endomorphin-1 affecting innate immune cells in vitro

Opioid peptides belong to one of most studied groups of regulatory peptides due to exerting extremely wide range of biological activities and play an important role in regulating homeostasis. The endogenous opioid system is involved in the functioning of diverse organs and systems, including the immune system. Currently, an immunomodulatory effect of the three major families of opioid peptides such as endorphins, enkephalins, dinorphins has been thoroughly described. Much less data are available about the endomorphin-related immunomodulatory effects. The aim of this work was to assess effects of endomorphin-1 on phagocytosis, production of reactive oxygen species and IL-1β by various subsets of peripheral blood innate immune cells in vitro. The leukocytes of obtained from peripheral venous blood of healthy volunteer donors aged 22 to 40 years were used in the study. Endomorphin-1 was used at concentrations of 10-6, 10-8, 10-10, 10-12 М. Blood leukocyte fraction was isolated from heparinized venous blood settled for 2 hours in a thermostat at 37 °С. The neutrophil fraction was isolated by centrifuging in of Ficoll-Urographin (ρ = 1.077) density gradient placing the upper layer of blood plasma with leukocytes. Assessment of leukocyte oxygen-dependent microbicidal activity was carried out by using the reaction of luminol-dependent chemiluminescence (LZHL) by using opsonized zymosan at concentration of 150 μg/ml; 10-5 M luminol was used to probe reaction magnitude. The separation of the mononuclear cell fraction was carried out by centrifuging in of Ficoll-Urographin (ρ = 1.077) density gradient. The monocyte fraction was isolated mechanically. To assess IL-1β production, mononuclear cells and monocytes were cultured for 24 hours followed by measuring its level with enzyme-linked immunosorbent assay. To evaluate monocytes and neutrophil phagocyte activity, FITC-labeled St.aureus uptake method was used. Statistical processing was performed with one-way ANOVA test and paired LSD criterion for post-hoc comparison, with significance set at p 0.05. It was found that endomorphin-1 reduced leukocyte spontaneous, but not induced production of reactive oxygen species. In the neutrophil fraction, endomorphin-1 did not affect spontaneous oxygen dependent microbicidity and reduced intensity of the respiratory burst in stimulated neutrophils. In addition, it increased the percentage of monocyte phagocytosis and enhanced spontaneous IL-1β production by mononuclear cells. Thus, endomorphin-1 exhibited multidirectional effects on various parameters of innate immunity. Being typical to other groups of regulatory peptides, modality of endomorphin-1 related effects depended on cell fraction and presence of a stimulating cues.

APPLICATION OF TIME-OF-FLIGHT MASS-SPECTROMETRY FOR DETECTION OF CAUSATIVE AGENT OF BRUCELLOSIS IN BLOOD SAMPLES IN EXPERIMENT

Aim.Study the possibility to apply time-of-flight mass-spectrometry for detection of causative agent of brucellosis in blood. Materials and methods. Brucella strains: 5 Brucella melitensis and 21 Brucella abortus. Protein profiling in linear mode on MALD1-TOF mass-spectrometer Microflex «Bruker Daltonics». Results. Technique for disinfection and preparation of blood samples was modified and optimized for MALDI-TOF MS analysis. 120 representative protein profiles of sera extract were obtained that contain brucellosis causative agent. A resulting peak-list (super-spectrum) of the studied protein fraction of blood extract of a conditionally healthy human within the studied group was formed and analyzed. Conclusion. A scheme of brucella detection in blood samples by MALDI-TOF MS is proposed, based on detection of a complex of 15 genus-specific fragments. Signals on mass-spectra of extracts of leukocyte fraction of blood, artificially contaminated with brucellosis causative agents are characterized.

Plasma contributes to the antimicrobial activity of whole blood against Mycobacterium tuberculosis.

The whole blood model for infection has proven useful to analyze the immunological response to Mycobacterium tuberculosis, because it exerts a significant antimicrobial activity. Although this activity has been generally assumed to be cellular, we have found that the leukocyte fraction of blood from healthy volunteers did not kill the bacilli. We have discovered that plasma was responsible for a large proportion, but not all, of the antimicrobial activity. Furthermore, infected monocytes controlled the mycobacterial multiplication when cultivated in the presence of plasma. Intriguingly, serum from the same donors did not share this activity, although it was able to eliminate the non-pathogenic Mycobacterium gordonae To identify the remaining components that participate in the antimycobacterial activity we fractionated blood in leukocytes, plasma, erythrocytes and platelets, and analyzed the bactericidal power of each fraction and their combinations using a factorial design. We found that erythrocytes, but not platelets, participated and showed by flow cytometry that mycobacteria physically associated with erythrocytes. We propose that in exposed healthy individuals that show 'early clearance' of the mycobacteria, the innate response is predominantly humoral, probably through the effect of antimicrobial peptides and proteins.

Abstract B28: Validation of a combined 13 gene six protein blood signature for earlier detection of ovarian cancer.

Abstract Epithelial ovarian cancer (EOC) is diagnosed in more than 70% of the cases in advanced FIGO stages which renders EOC the most deadly gynecologic cancer. Up to date there are no specific symptoms and no screening tests for general population or for patients at risk. The development of early diagnostic tools that will improve clinical outcome and differentiate between benign gynecological diseases and EOC are still an unmet need. In a multicenter study financed by the Seventh Framework Program of the European Commission (TRANSCAN) called ImPECT, three European EOC centers (Medical University of Vienna, Charité in Berlin, and the European Institute of Oncology in Milan) validated a “combined blood based gene expression and plasma protein abundance signature for diagnosis of epithelial ovarian cancer” (Pils et al. BMC Cancer, 13:178). In total, blood of 576 women were enrolled in this validation study, including healthy controls, patients with EOC, with breast cancer (to assess tumor specificity), with benign gynecologic diseases (to assess the potential for differential diagnosis), and with inflammatory diseases and diabetes (to assess non-malignant immune changes). A specific blood leukocyte fraction and plasma were isolated and RNA prepared. The expression of 23 genes (13 from the diagnostic signature, seven from an unpublished prognostic signature and three house-keeping) were measured by a multiplexed hybridization based method (Nanostring's nCounter Elements™) and the abundance of six proteins from plasma by a multiplexed bead-based technology (Luminex). Due to evidence for substantial fluctuations of gene expression values by RT-qPCR, especially of the reverse transcription (RT) reaction, we replaced the RT-qPCR method by a hybridization based method without RT. Also, one protein of the protein panel (IGF2) was replaced by another protein (HE4) for two reasons: measurement of IGF2 cannot be multiplexed with the other proteins and HE4 was shown to have a higher discriminative power compared to IGF2. In a first step the published discriminative models were validated with 96 healthy controls and 96 EOC patients. All models showed significant area under the receiver operating characteristic (ROC) curve (AUC) values (p<0.05): Protein models, 0.951 (three proteins) and 0.954 (five proteins), gene expression models, 0.653 (seven genes) and 0.605 (13 genes), and combined models, 0.709 (four proteins/five genes) and 0.773 (five proteins/13 genes). The low AUC values, especially for the combined models, are disillusioning but probably due to the different measurement methods. Therefore new models were built using all 20 gene expression and six protein abundance values using more robust machine learning approaches, namely penalized Support Vector Machines (SVMs). Indeed, much better discriminative models could be built, discriminating healthy controls from EOC patients: Five proteins, AUC 0.972 (CI95:0.952-0.993); 16 genes, AUC 0.848 (CI95:0.794-0.903), and four proteins/five genes (combined signature), AUC 0.981 (CI95:0.963-0.998). The last, combined, signature is significantly better compared to the first, only protein, signature (p=0.029), indicating an additional discriminative value for the gene expressions. To assess the technical reproducibility of the Nanostring method, each 84 samples were measured a second time and correlated to the first measurement: Interestingly, the median Pearson's r correlations over all 20 genes was only 0.689 (0.337-0.799) but the SVM model between controls and EOC patients was more robust: AUC of 0.811 compared to 0.848 (p=0.394). A clinical validation of these models with independent patient and control cohorts (healthy controls and patients with benign diseases) and the specificity compared to breast cancer and diseases with immune system involvement will be presented. Citation Format: Anna Bachmayr-Heyda, Stefanie Aust, Katharina Auer, Peter Borossay, Nina Pecha, Ugo Cavallaro, Ioana Braicu, Sarah Igidbashian, Costanza Savino, Franchi Dorella, Jalid Sehouli, Sonia Zaafrani, Florian Fitzal, Christian Singer, Michael Gnant, Robert Zeillinger, Dietmar Pils. Validation of a combined 13 gene six protein blood signature for earlier detection of ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: Exploiting Vulnerabilities; Oct 17-20, 2015; Orlando, FL. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(2 Suppl):Abstract nr B28.

The Dynamics of the Epidemiological Situation of Tick Borne Encephalitis in the Far East

We have presented a comparative analysis of morbidity and mortality in the tick-borne encephalitis (TBE) in the south of the Russian Far East in the period 1980 - 2014. In the period from 2008 to 2014 using the enzyme-linked immunoassay (ELISA) was examined 10,599 copies ticks or leukocyte fraction of blood (n = 5561) of people who indicate to the fact of tick bites. Blood samples and ticks positive in ELISA additionally examined by PCR and virus isolation. Under diagnosis of TBE cases in the 1980s resulted in lower figures morbidity and mortality artificially excessive to 39% (average 28%). In the 1990s there was a sharp rise in the incidence of TBE (median 15.3% mortality). In the 2000s there was a trend to a decrease in the number of cases and deaths (average case fatality rate of 6.3 -10.5%). The incidence rate for 35-year follow-up period ranged from 0.85 to 9.88 per 100 thousand population, mortality was 14.8 ± 0.7%. The degree of danger of the epidemic on TBE synchronously reflected in the rate of antigen detection of TBE virus in humans and ticks attached themselves. In the year’s high morbidity and mortality (in 2009 and 2010) antigen detection in ticks and in human blood reached a maximum. Isolated from the blood of these patients three TBE virus strains indicated that only a complete virus capable of replicating determines the variety clinical forms of the infection.

Acute Stress Elicited by Bungee Jumping Suppresses Human Innate Immunity

Although a relation between diminished human immunity and stress is well recognized both within the general public and the scientific literature, the molecular mechanisms by which stress alters immunity remain poorly understood. We explored a novel model for acute human stress involving volunteers performing a first-time bungee jump from an altitude of 60 m and exploited this model to characterize the effects of acute stress in the peripheral blood compartment. Twenty volunteers were included in the study; half of this group was pretreated for 3 d with the β-receptor blocking agent propranolol. Blood was drawn 2 h before, right before, immediately after and 2 h after the jump. Plasma catecholamine and cortisol levels increased significantly during jumping, which was accompanied by significantly reduced ex vivo inducibility of proinflammatory cytokines as well as activation of coagulation and vascular endothelium. Kinome profiles obtained from the peripheral blood leukocyte fraction contained a strong noncanonical glucocorticoid receptor signal transduction signature after jumping. In apparent agreement, jumping down-regulated Lck/Fyn and cellular innate immune effector function (phagocytosis). Pretreatment of volunteers with propranolol abolished the effects of jumping on coagulation and endothelial activation but left the inhibitory effects on innate immune function intact. Taken together, these results indicate that bungee jumping leads to a catecholamine-independent immune suppressive phenotype and implicate noncanonical glucocorticoid receptor signal transduction as a major pathway linking human stress to impaired functioning of the human innate immune system.

From leukocyte reduction to leukocyte transfusion: the immunological effects of transfused leukocytes

In transfusion medicine, mononuclear leukocytes have been studied more often as contaminants of red blood cells or platelets responsible for adverse transfusion outcomes than as therapeutic cells; leukocyte transfusion has been effective in augmenting recipient immunity only in limited clinical situations. Studies in leukocyte reduction and leukocyte transfusion have progressed separately as if the leukocytes' adverse and therapeutic effects result from different immunological mechanisms. With growing clinical experience, however, it is increasingly clear that some adverse immune effects may be exploited for therapeutic benefit. Advances in clinical immunology, understanding of the variety of cells and functions in the leukocyte fraction of blood, and blood component preparation technology may lead to new ways of deriving immunological benefit from transfused blood leukocytes while minimizing their adverse effects. This chapter reviews the current uses of leukocyte reduction and mononuclear leukocyte transfusion, with an emphasis on the relationship between transfusion-associated graft-versus-host disease and donor lymphocyte infusion in controlling relapsed leukaemias.

Ultrastructural and antigenic characterization of a granulocytic ehrlichiosis agent directly isolated and stably cultivated from a patient in New York state.

A human granulocytic ehrlichiosis (HGE) agent with 16S rDNA sequence identical to the published sequence of HGE agents was isolated from a patient from New York State by inoculation of the blood leukocyte fraction directly into a human promyelocytic leukemia cell line HL-60. The HGE agent was also isolated from the leukocyte fraction of the blood and bone marrow of a mouse inoculated with the leukocyte fraction of the patient's blood. The isolate has been passaged in tissue culture 30 times over 8 months. Electron microscopy revealed pleomorphic coccobacilli with a thin and highly rippled outer membrane in the clear inclusion matrix. Comparison of IFA reactivity of antisera obtained from a variety of sources with the cell-cultured HGE agent revealed that 3 HGE agent strains (New York isolate, Wisconsin [BDS] isolate, and a tick-derived isolate) are highly cross-reactive and there are diverse antigenic cross-reactivities between HGE agent and Ehrlichia chaffeensis.

Lymphocyte subset responses to repeated submaximal exercise in men

The effects of repeated bouts of submaximal cycle ergometry exercise on changes in the percentage of peripheral blood T-lymphocytes, the T-helper/inducer and T-cytotoxic/suppressor subsets, and natural killer (NK) cells were studied in 18 healthy young men who had no history of regular exercise training. Subjects were matched on the basis of maximal O2 uptake and assigned randomly to exercise or control groups, with controls resting quietly during the exercise sessions. The percentage of peripheral blood mononuclear leukocytes that reacted with monoclonal antibodies specific for T-lymphocytes (CD3+ cells), the helper/inducer subset (CD4+ cells) and cytotoxic/suppressor subset (CD8+ cells) of T-lymphocytes, and cells with NK activity (Leu7+ cells) were enumerated by fluorescence-activated flow cytometry for samples obtained immediately before and after exercise on days 1, 3, and 5 of a 5-day exercise regimen. The results of this study were mixed with decreases in the percentage of T-lymphocytes before vs. after exercise on days 1 and 3 (P less than 0.001), a decrease in the percentage of T-helper/inducer cells before vs. after exercise on day 3 (P less than 0.05), no effect of exercise on the percentage of T-cytotoxic/suppressor cells, and a marked increase in the percentage of NK cells after exercise on days 1 (P less than 0.05) and 3 (P less than 0.01). The total number of recovered NK cells in the mononuclear leukocyte fraction of blood also increased significantly after exercise on days 1 (P less than 0.05) and 3 (P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Causative ehrlichial organisms in Potomac horse fever.

An ehrlichia was consistently isolated from the peripheral blood leukocyte fraction of ponies that had been experimentally infected with Potomac horse fever by whole blood transfusion from naturally infected horses. The organism was propagated in a human histiocyte cell line for 3 to 5 weeks and then inoculated intravenously or intradermally into healthy adult ponies. Clinical signs of Potomac horse fever, which varied in the degree of severity, occurred 9 to 14 days post-inoculation in all of the ponies. One pony died 20 days post-inoculation. The ehrlichial organism was reisolated in the human histiocyte cell line from the blood leukocyte fraction of all of the experimental ponies on each day that samples were examined (days 9, 10, 11, 19, and 39). These organisms were identical to those originally detected in the wall of the intestine of ponies with clinically diagnosed Potomac horse fever when compared by light and electron microscopy and an immunofluorescence labeling technique. The immunofluorescent antibody titer became positive in a pony at 20 days postinjection. These results indicate that the ehrlichial organisms is the causative agent of Potomac horse fever.

Prediction of survival in head and neck cancer based on leukocyte sedimentation in ficoll‐hypaque gradients

A battery of immunologic tests were performed on 42 patients with recurrent cancer of the head and neck whose median survival time was 6.8 months. Two tests correlated with survival: total white blood cell (WBC) count and the percentage of lymphoid cells (%LG) in the Ficoll-Hypaque gradient separated leukocyte fraction of blood. Patients with abnormally low values of %LB had a poor prognosis, as did patients who had greater than 8000 WBC count. Of the variation in %LG, 43% was due to the differences in differential WBC counts among the patients, with the remaining variation attributed to changes in the buoyant densities of the leukocytes. These data suggest that further study of the factors effecting buoyant density of cells may be clinically useful.

Serum stimulation of phospholipase A2 and prostaglandin release in 3T3 cells is associated with platelet-derived growth-promoting activity.

Sera from mouse, rat, and calf sources stimulate cellular phospholipase A2 activity (PLase; phosphatide 2-acylhydrolase, EC 3.1.1.4) and prostaglandin synthesis in 3T3 Swiss mouse fibroblasts, releasing up to 33% of biosynthetically incorporated [3H]arachidonic acid as hydrolysis products in 1 hr. The PLase stimulated by mouse serum exhibits specificity for arachidonic acid residues on phospholipids. It is stimulated 2.5-fold by 1.8 mM Ca2+ in the presence of 5 microM divalent cation ionophore A23187, consistent with a Ca2+-dependent enzyme possessing a cytoplasmic Ca2+ binding site. The percentage maximal PLase- and growth-stimulating activities of the three sera exhibit similar concentration dependencies, with the homologous (mouse) serum exhibiting the highest specific activities. Confluent 3T3 cells deplete PLase-stimulating activity from medium containing calf serum at a rate similar to the depletion of cell growth-stimulus activity. The PLase-stimulating activity in rat and mouse sera is derived from the leukocyte fraction of blood, presumably from platelets. In rat leukocyte lysates the PLase-stimulating activity exhibits properties similar to those reported for platelet-derived cell growth factors from rat and human sources--i.e., stability to exposure to 100 degrees C for 2 min or to pH 2 or pH 11, and cationic properties. Purified preparations of human platelet-derived growth factor also exhibit 3T3 PLase-stimulating activity.

Membrane immunoglobulins of bovine leukemic lymphocytes

Membrane immunoglobulins were isolated from B lymphocytes of the blood leukocyte fraction of a leukemic cow. The immunoglobulin was serologically of IgM class but its μ chain was bigger than that of IgM from normal bovine serum or IgM from the serum of the leukemic cow. The difference in apparent molecular weight between the μ chain of the membrane immunoglobulin and that of secreted IgM was of approximately 10,000 daltons. The possible reasons for this observation are discussed.