Group Of Healthy Donors Research Articles

The in vitro Mycobacterium bovis\xa0BCG Moreau infection of human monocytes that induces Caspase-1 expression, release and dependent cell death is mostly reliant upon cell integrity

BackgroundCaused by Mycobacterium tuberculosis, tuberculosis (TB) is an extremely contagious disease predominantly affecting the lungs. TB is found worldwide and has a major impact on public health safety primarily due to its high mortality rate. Applied for over a hundred years as a preventive measure, Mycobacterium bovis BCG remains the only available TB vaccine. Only one seminal study about the apoptotic pathways induced by this vaccine in the monocytic lineage of the host cell has found the effects of BCG on regulation of apoptosis. The aim of this study was to explore beyond that pioneer study the pathway related to the in vitro cell-death pattern and the inflammatory response to the BCG vaccine in human monocytes.MethodsCohorts of HIV-negative volunteers were enrolled: adult Healthy Donors (HD) and neonates’ Umbilical Cord Blood (UCB) individuals. Host mononuclear cells were infected with the M. bovis Moreau strain of BCG vaccine at 16, 24, 48, and 72 h. The Real-Time RT-PCR for TRADD, Bcl-2, and Caspases-1 and -3 were performed, and supernatants were assayed in parallel for Caspase-1, NLRP3, HO-1, and IL-1β levels whereas caspases were assessed intracellularly. The effect of a BCG infection in monocytes was characterized via a metabolic activity assay by LDH release profiles.ResultsOverall, the BCG vaccine induced significantly higher Caspase-1 and Bcl-2 mRNA levels in both the HD and UCB groups (p-value ≤0.05). In addition, a significant increase solely in Caspase-1 protein levels was also noted in both HD and UCB (p-value ≤0.05) notwithstanding the absence of any damaged cell membranes.ConclusionsOur data directly corroborate other findings showing that BCG Moreau led to an increased secretion of IL-1β but not IL-18, two Caspase-1-activated cytokines, and are also in support of the model that the BCG Moreau infection of human mononuclear cells may induce a cell-death pattern involving Caspase-1 activation.

Cell-free DNA analysis in healthy individuals by next-generation sequencing: a proof of concept and technical validation study

Pre-symptomatic screening of genetic alterations might help identify subpopulations of individuals that could enter into early access prevention programs. Since liquid biopsy is minimally invasive it can be used for longitudinal studies in healthy volunteers to monitor events of progression from normal tissue to pre-cancerous and cancerous condition. Yet, cell-free DNA (cfDNA) analysis in healthy individuals comes with substantial challenges such as the lack of large cohort studies addressing the impact of mutations in healthy individuals or the low abundance of cfDNA in plasma. In this study, we aimed to investigate the technical feasibility of cfDNA analysis in a collection of 114 clinically healthy individuals. We first addressed the impact of pre-analytical factors such as cfDNA yield and quality on sequencing performance and compared healthy to cancer donor samples. We then confirmed the validity of our testing strategy by evaluating the mutational status concordance in matched tissue and plasma specimens collected from cancer patients. Finally, we screened our group of healthy donors for genetic alterations, comparing individuals who did not develop any tumor to patients who developed either a benign neoplasm or cancer during 1–10 years of follow-up time. To conclude, we have established a rapid and reliable liquid biopsy workflow that allowed us to study genomic alterations with a limit of detection as low as 0.08% of variant allelic frequency in healthy individuals. We detected pathogenic cancer mutations in four healthy donors that later developed a benign neoplasm or invasive breast cancer up to 10 years after blood collection. Even though larger prospective studies are needed to address the specificity and sensitivity of liquid biopsy as a clinical tool for early cancer detection, systematic screening of healthy individuals will help understanding early events of tumor formation.

High Concentrations of Serum Soluble E-Cadherin in Patients With Q Fever.

Cadherins switching is a hallmark of neoplasic processes. The E-cadherin (E-cad) subtype is one of the surface molecules regulating cell-to-cell adhesion. After its cleavage by sheddases, a soluble fragment (sE-cad) is released that has been identified as a pro-carcinogenic inflammatory signal in several bacteria-induced cancers. Recently we reported that Q fever, a disease due to Coxiella burnetii infection, can be complicated by occurrence of non-Hodgkin lymphoma (NHL). Therefore, we studied E-cad switching in Q fever. The sE-cad levels were found increased in the sera of acute and persistent Q fever patients, whereas they remained at the baseline in controls groups of healthy donors, people cured of Q fever, patients suffering from unrelated inflammatory diseases, and past Q fever patients who developed NHL. These results indicate that sE-cad can be considered as a new biomarker of C. burnetii infection rather than a marker of NHL-associated to Q fever. We wondered if changes in sE-cad reflected variations in the CDH1 gene transcription. The expression of E-cad mRNA and its intracellular ligand β-catenin was down-regulated in peripheral blood mononuclear cells (PBMCs) of patients with either acute or persistent forms of Q fever. Indeed, a lower cell-surface expression of E-cad was measured in a minority (<5%) subpopulation of HLADR+/CD16+ monocytes from patients with acute Q fever. However, a very strong increase in E-cad expression was observed on more than 30% of the HLADR+/CD16+ monocytes of persistent Q fever patients, a cell subpopulation known to be a target for C. burnetii in humans. An experimental in vitro infection of healthy donors' PBMCs with C. burnetii, was performed to directly evaluate the link between C. burnetii interaction with PBMCs and their E-cad expression. A significant increase in the percentage of HLADR+/CD16+ monocytes expressing E-cad was measured after PBMCs had been incubated for 8 h with C. burnetii Nine Mile strain. Altogether, these data demonstrate that C. burnetii severely impairs the E-cad expression in circulating cells of Q fever patients.

Update of reference values for IgG antibodies against typical antigens of hypersensitivity pneumonitis

Specific (s)IgG antibodies against environmental and occupational antigens, especially from bacteria, moulds, yeasts, birds and chemicals play an important role for hypersensitivity pneumonitis (HP). An increased serum level of sIgG is one criterion in the diagnostic procedure of HP and crucial for the detection of the triggering antigen for successful avoidance of further exposure. In contrast to specific IgE, sIgG concentrations in healthy individuals vary greatly depending on the antigen, which makes it difficult to differentiate from patients with HP. The aim of this study is to update or establish sIgG-reference values for important HP antigens in a healthy blood donor group. Therefore a study including six clinical centres in Germany was conducted to collect sera from 121 subjects without any signs of HP and without obvious exposure to potential HP antigens. Specific IgG to 32 typical HP antigens were quantified by ImmunoCAP (ThermoFisher Scientific; Phadia, Uppsala, Sweden). For validation selected measurements were repeated, total IgG was determined, sera were tested for unspecific binding with the human serum albumin ImmunoCAP Ro401, and influence of potential confounders was analysed. Statistical distribution of the antigen-specific IgG values was evaluated and the nonparametric method of percentile calculation was applied. The levels of IgG antibodies to the different antigens varied considerably in the study group from <0.02 to 726 mgA/L. Low sIgG levels were found against the chemicals and the highest levels to fungal antigens, especially to Aspergillus fumigatus and Botrytis cinerea. For three isocyanates, three acid anhydrides, Trichosporon pullulans and Acremonium kiliense reference values were proposed for the first time. For several avian antigens, moulds, and bacteria pre-existing reference values nearly could be confirmed without significant deviations, but already the 90% quantile for sIgG against Penicillium chrysogenum, Aspergillus fumigatus and pigeon antigen (Ge91) clearly exceeded the pre-existing values. In contrast, the 97.5% quantile value for Candida albicans was nearly half of the pre-existing cut-off value. In most cases specific IgG values were not significantly influenced by smoking and gender and most of them were unaffected by age. For implementation of these sIgG reference values into the routine diagnosis of HP, we provide an online available calculator to rank measured sIgG concentrations to the 32 different ImmunoCAP antigens.

Subpopulations of blood monocytes in patients with generalized hypoxia

The aimof the work is to establish general regularities and features of differentiation of blood monocytes into 4 subpopulations in diseases associated with circulatory and respiratory hypoxia.Materials and methods.18 patients with ischemic heart disease (IHD), 12 patients with ischemic cardiomyopathy (ICMP), 14 patients with chronic obstructive pulmonary disease (COPD), 15 patients with newly diagnosed infiltrative pulmonary tuberculosis (PTB) and 12 healthy donors were examined. In whole blood, we determined the relative number of different subpopulations of monocytes by flow cytometry. The results were analyzed by statistical methods.Results.It is shown that an increase in the number of classical (80.56 [77.60; 83.55]%) and the deficit of intermediate (10.38 [9.36; 11.26]%), non-classical (6.03 [5.24; 6.77]%) and transitional (2.14 [1.41; 3.92] %) monocytes in the blood is determined in patients with COPD when compared with the group of healthy donors (p&lt; 0.05). In groups of patients with PTB and IHD, an increase in the number of intermediate monocytes (26.24 respectively [22.38; 42.88] % and 25.27 [15.78; 31.39]%) and the lack of transitional cells (1.77 [1.36; 3.74]% and 2.68 [2.63; 4.0]%) at the normal content of classical and non-classical forms of monocytes (p&lt; 0.05) is detected. In patients with ICMP, a decrease in the number of non-classical monocytes (up to 5.05 [4.08; 6.58]%) is combined with the normal cell content of other subpopulations (p&lt; 0.05). The interrelation between the number of classical and intermediate monocytes in patients with COPD (r= –0.63;p&lt; 0.05), PTB (r= –0.72;p&lt; 0.01), IHD (r= –0.59;p&lt; 0.05), ICMP (r= –0.58;p&lt; 0.05) was established.Conclusion.In COPD associated with generalized hypoxia, an increase in the number of classical monocytes is combined with a deficiency of their other subpopulations in the blood. In PTB and IHD, antigenic stimulation of the immune system mediates accelerated differentiation of monocytes from classical to intermediate forms with a decrease in the number of transitional cells regardless of the etiology of the disease (infectious or non-infectious) and the type of hypoxia (respiratory or circulatory).

Molecular characterization and antibiotic sensitivity testing of bacteria in blood cultures of Hepatitis B virus infected subjects

Introduction: Hepatitis B virus is one of the most common infections worldwide. Many infected people are at risk of developing liver complications. Screening for common pathogenic bacterial infections that could contribute to complications is important for early diagnosis and appropriate management. Methods: A cross sectional study was carried out on subjects aged 20-75 years for a period of 6 months (November 2016 to April 2017). Blood cultures and HBsAg rapid tests were performed on all 122 blood samples collected in Ilorin Metropolis. The screening was carried out on 92 HbsAg positive patients who presented with fever, and 30 apparently healthy HbsAg positive donors from the blood bank. Results: Of 92 symptomatic patients, 44 (47.8%) had postive blood cultures and of the 30 HBV positive blood donors, 9 (30%) had positive blood cultures. The prevalence of Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa and Klebsiella pneumoniae in Hepatitis B positive subjects was 5.7% (n=7), 5.7% (n=7), 23.8% (n=29) and 9.8% (n=12) respectively. In the apparently healthy HbsAg positive blood donor group, only 9 samples showed positive bacterial growth of P. aeruginosa. All the bacterial isolates were resistant to amoxycillin-clavulanic acid, erythromycin, ceftriaxone, ceftazidime, cefuroxime, and ciprofloxacin. On PCR, Nuc, Stx2, Pf and PSUE genes were demostrated in E. coli, S. aureus, P. aeruginosa and K. pneumoniae respectively. Conclusion: This study showed a high percentage (45.1%) of bacteraemia in HBV infection. Early screening and treatment of HBV infection and concomitant bacterial infection is recommended to prevent complications.

The frequency of Merkel cell polyomavirus in whole blood from immunocompetent and immunosuppressed patients with kidney disease and healthy donors

Merkel cell polyomavirus (MCPyV) is a rare, aggressive and related to human diseases in immunocompromised patients. MCPyV has been detected in skin neoplasms, various cancers, immunosuppressed patients and immunocompetent individuals. Several studies have confirmed the presence of MCPyV in patients with kidney dysfunction, such as kidney transplant (KTx) and long-term dialysis patients.The aims of this study were to quantify and compare the frequency of MCPyV in whole blood samples from immunocompetent and immunosuppressed patients and healthy blood donors and to compare MCPyV genotypes in a Korean population.DNA from Groups 1, 2, and 3 was screened for MCPyV using polymerase chain reaction (PCR) and quantitative real-time PCR (qPCR) with primer pairs targeting two regions of the large T-antigen.Thirteen of 122 whole-blood samples (12.7%) were positive for MCPyV. The virus was detected in the three groups of patients and healthy donors; specifically, in 5 of 30 (16.7%) KTx patients (Group 1), 6 of 52 (11.5%) dialysis patients (Group 2), and 4 of 40 (10%) healthy donors (Group 3). Low viral DNA loads 4.4–18 copies/μl were observed using qPCR DNA sequences from the two MCPyV-LT regions, which showed high homology with MCPyV sequences belonging to the TKS strain from Japan rather than the Chinese/European/North American strains. The MCPyV DNA was similarly amplified in whole blood from immunocompetent and immunosuppressed patients and healthy donors. This virus may be involved in establishing the persistence of infected peripheral leukocytes in the host, based on the incidence of detection of MCPyV DNA in blood samples from immunocompromised and immunocompetent subjects. This study is the first to identify a Korean MCPyV strain in whole-blood samples from Korean patients with kidney disease and healthy individuals.

Features of biochemical changes in patients with localized scleroderмa

The aim of the study was to determine the characteristics of biochemical changes in localized scleroderma based on the analysis and evaluation of dysmetabolic disorders of homeostasis from the standpoint of the individual characteristics of patients.Materials and methods. Under our supervision there were 107 patients with localized scleroderma in the acute stage who were hospitalized in the Department of Dermatology of the State Institution “Institute of Dermatology and Venereology of the National Academy of Medical Sciences of Ukraine” from 2014 to 2018. All patients were randomly assigned to 3 groups comparable in all parameters — the main group and two comparison groups. The main group consisted of 33 (34.01 %) localized scleroderma patients who received complex treatment; in the І group - 34 (35.05 %) patients who received only traditional therapy; in group II - 30 (30.92 %) patients who received treatment according to the scheme, which included traditional therapy with the addition of thiotriazoline.The study of the concentration of C-reactive protein (CRP) was performed by quantitative immunoturbidimetry according to OP. Shevchenko (1997); serum protein fractions - by electrophoresis on agarose gel plates by the method of S.S. Chernecky, V.J. Berger (2008); total lipids - by V.N. Titovu, M.G. Tvorogova (1992); high-density lipoproteins, triglycerides, beta-lipoproteins - by S.E. Severin (1977);cholesterol concentration - according to D. Vaidya (2005); the atherogenic coefficient was calculated by the formula: CA = (total cholesterol - HDL) / HDL); determination of serum magnesium and chlorine using colorimetric analysis (A.Sh. Byshevsky, OA Tersenov, 1994).Results. In the studied groups of patients with OSD and in healthy donors, a study was made of the level of C-reactive protein (CRP., The concentration of which in patients with OSD in the exacerbation stage reached 44 g / l before treatment, after conducting a course of appropriate drug therapy in each study group amounted to: in group II, the comparison group — 38 g / l, in group I, the comparison group — 30 g / l, and in the main group — 16 g / l, which are closest to the level of CRP in healthy donors and indicate high efficacy of the treatment we developed a scheme with the inclusion of modern drugs.In the serum of patients of the I and II comparison groups, there was an increase in magnesium content by 1.8 times and a decrease in chlorine level by 1.2 times, which characterizes the presence of deep metabolic disorders in this category of patients, affecting not only immunoreactivity but also water-electrolyte metabolism. The magnesium content and the level of chlorine in the blood serum of the patients of the main group did not significantly differ from the control and was 2 times lower in comparison with the concentration of these indicators in the group of patients with SJD before treatmentConcentration of cholesterol was lower in the OSD group before treatment and in the comparison groups - an average of 1.8 times. Also noted increase in the concentration of high density lipoprotein - an average of 2.7 times. The concentration of triglycerides was minimal in the CSF group before treatment and was 0.4 ± 0.03 mol / L, which was 5.4 times lower in the control group of healthy donors and 4 times lower than in the main group of patients.Conclusion. Based on the conducted research, a number of features of biochemical parameters were revealed, which indicate that localized scleroderma is accompanied by disturbances of the biochemical link of homeostasis. The change in the ratio of lipid and protein fractions indicates a violation of the regulatory and regenerative functions at the level of the structural organization of cell membranes, and an increase in the magnesium content and a decrease in the level of chlorine — deep metabolic disorders in this category of patients

The Significance of Serum S100A9 and TNC Levels as Biomarkers in Colorectal Cancer.

Purpose: The aim of this study was to evaluate the diagnostic value of S100A9 and tenascin-c (TNC) levels as colorectal cancer (CRC) biomarkers in several ways, including through screening tests, differentiation tests, combination with existing biomarkers (CEA and CA19-9), and serum level measurements before and after surgery.Materials and Methods: In this case-control study, S100A9 and TNC serum levels were measured in 460 participants: 258 CRC patients, 99 patients with benign colonic disease (BCD) and 103 healthy donors (HD).Results: The serum levels of S100A9 were 22.32 (14.88-29.55) ng/ml, 10.02 (5.83-14.15) ng/ml and 10.05 (7.68-15.34) ng/ml in the CRC, BCD and HD groups, respectively. The serum levels of TNC were 4.30 (2.12-6.04) ng/ml, 1.60 (1.06-2.30) ng/ml and 2.00 (1.37-3.00) ng/ml in the CRC, BCD and HD groups, respectively. Significantly higher levels of both biomarkers (S100A9 and TNC) were found in CRC patients (both p<0.001).Both S100A9 and TNC levels were superior to CEA and CA19-9 levels as CRC diagnostic biomarkers; the combination of S100A9, TNC and CEA levels was an excellent biomarker with 79.8% sensitivity and 89.6% specificity. The serum levels of S100A9 and TNC in CRC patients were significantly lower after surgery than before surgery (p<0.01).Conclusion: S100A9 and TNC levels could serve as diagnostic biomarkers of colorectal cancer.

Analysis of factors that may influence the methylation pattern of oral mucosa

Analysis of factors that may influence the methylation pattern of oral mucosa

Using cytometric bead arrays to detect cytokines in the serum of patients with different types of pulmonary tuberculosis.

Cytokines play a crucial role in mediating immune responses to tuberculosis (TB). The aim of this study was to evaluate the levels of cytokines in patients with different forms of pulmonary tuberculosis (PTB) and identify valuable cytokine biomarkers for the diagnosis of PTB. We measured the levels of six cytokines (interleukin (IL-2, IL-4, IL-6, and IL-10), tumor necrosis factor (TNF-α), and interferon-γ (IFN-γ)) in the serum of healthy donors (n = 30). Patients with active PTB (n = 46) and those with latent tuberculosis infection (LTBI, n = 38) were examined using cytometric bead arrays. The levels of the six cytokines in the serum samples were measured promptly, sensitively, and simultaneously. The levels of IL-2, IL-6, IL-10, and IFN-γ were significantly higher in the PTB group compared with those reported in the healthy donors (P < 0.01 or P < 0.05). In addition, significantly higher levels of IL-2, IL-6, IL-10, and IFN-γ were found in the active PTB group compared with those observed in the LTBI group (P < 0.01 or P < 0.05). However, the levels of IL-4 and TNF-α in the sera of patients from the PTB group did not show a significant correlation with those measured in the healthy donor group. Our data demonstrated that IL-2, IL-6, IL-10, and IFN-γ may be useful in the auxiliary diagnosis of tuberculosis and as biomarkers for distinguishing LTBI from TB.

Assessment of Postvaccine Immunity against Streptococcus pneumoniae in Patients with Asplenia, including an Analysis of Its Impact on Bacterial Flora of the Upper Respiratory Tract and Incidence of Infections.

S. pneumoniae is a microorganism that may cause a serious threat in postsplenectomy patients due to a potentially invasive course of infection. In order to assess a protective activity after vaccination with the 23-valent vaccine, we made an analysis of the level of antibodies in patients with asplenia compared to a control group of healthy donors. Additionally, colonization by potentially pathogenic microorganisms of the upper respiratory tract was analyzed to determine the carrier state by strains with vaccine serotype. No such strains were found in the research, yet three non-vaccine-serotype strains were found. Colonization of the upper respiratory tract by potentially pathogenic microorganisms may be connected with increased susceptibility observed and incidence of infections in patients with asplenia. However, colonization by S. pneumoniae may not have an effect on the level of specific antibodies with the 23-valent vaccine against S. pneumoniae (PPV23) in postsplenectomy patients and healthy people. The response to vaccination against S. pneumoniae showed a lower level of specific antibodies in patients with splenectomy performed more than 2 years before the test than in patients with a recently removed spleen, i.e., from 1 month to 2 years before the test. Vaccination against pneumococci also has positive effects on incidence of other etiology infections, which is of high significance in the prophylaxis of infectious diseases in this group of patients.

Characterization of Circulating and Bone Marrow Derived Exosomes in Multiple Myeloma Patients

Background: Multiple myeloma (MM) is characterized by clinical and biological heterogeneity, translating to variable outcome. High International Staging System (ISS), high risk cytogenetics, elevated LDH, poor response to novel drugs or extramedullary-disease (EMD) are associated to poor prognosis. Exosomes (EXO) are extra vesicular particles implicated in intercellular communication. Exosomal cargo has recently been studied as prognostic factors in cancer. MM-EXO may act as biomarkers of disease aggressiveness and potentially be used for better stratification of high risk disease in the clinical setting as liquid biopsies.Aims: To characterize peripheral blood (PB) and bone marrow aspirates (BM) EXO in MM and MGUS patients versus healthy donors (HD) and to analyze its potential use as a prognostic biomarker.Methods: EXO were isolated from PB and BM samples by ultracentrifugation. EXO protein quantity (EXOcargo) was determined by the ratio between total quantity of proteins and the total number of exosomes. Comparisons among groups of patients (MM, MGUS) and HD were made regarding EXO total protein cargo (PB and BM).Results: MM patients revealed 5x times higher EXOcargo in PB (ug/EXO) when compared to MGUS patients (p=0.007, n=15 MM and n=8 MGUS); MM median 2.34 (1.08-6.5) ug/EXO versus MGUS median 0.51 (0.29-1.04) ug/EXO. There was no differences regarding the EXOcargo between the MGUS and HD (n=6, HD median 0.88 (0.57-1.83)ug/EXO). In BM, MM EXOcargo was also higher than in MGUS: MM 2.09 (0.49-3.85)ug/EXO versus MGUS 0.53 (0.21-0.831)ug/EXO. Analyzing the patients with higher EXOcargo, we found that patients with EMD (n=4) are among the ones with higher circulating exosomal protein cargo (median 5.76 (4.36-7.95). A positive correlation in EXOcargo between PB and BM in MM and MGUS (Spearman r=0.85; p< 0.0001) was found. We also studied circulating EXOcargo at response evaluation (T2) compared to EXOcargo before MM treatment (T1): patients with deeper response had significant lower EXOcargo (CR T2 median 0.85 (0.65-1.35) p<0,05); patients in VGPR (T2 median 1.52 (0.86-2.34) p=0.36) PR (T2 median 2.68 (2.03-3.33) p=0.94) and progression (T2 median 2.22 (1.0-2.46) p=0.58).Conclusion: 1-The positive correlation of the exosomal protein cargo in PB and BM samples may be a reflection of the BM compartment and potentially supports the use of EXO as liquid biopsies for biomarkers of BM disease, in a less invasive method. 2-MM patients have higher circulating exosomal protein cargo in PB when compared to MGUS patients and HD. 3- Patients with extramedullary disease and more aggressive forms of multiple myeloma have a higher exosomal cargo in the PB and this might be related to different biological features of the disease. The longitudinal evaluation in 2 different time-points suggests a correlation between EXO protein cargo and response to treatment, to be confirmed in a larger cohort. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Oligoclonal Expansion and T-Cells Subpopulations in Patients with Aplastic Anemia at Different Stages of the Disease

Background. The main mechanism of the bone marrow (BM) failure in idiopathic aplastic anemia (AA) has an immunomediated character. Researching the T-cell clone's effect in the AA pathogenesis is very relevant at the present time. Oligoclonal expansion of T cells is frequent in AA and the identification of immunodominant T-cell clones can correlate with the disease activity and may possibly serve as response predictor to immunosuppressive therapy (IST).The aim. To identify T-cells subpopulations, expression of PD-1 and PD-L1 on T-cells and TCR-Vβ repertoires by flow cytometry in different groups of AA patients.Methods. Thirty AA patients (pts) with median age of 30.5 (19-71), m/f ratio 1:1,3 were divided in 3 groups: pts with newly diagnosed (ND) AA (n=13), pts with overall response to IST (OR) (n=10), non-response pts (NR) for 2 and more lines of IST (n=7). Flow cytometry was performed with BD FACS Canto II. We used commercial kit (IOTest® Beta Mark TCR Vb Repertoire) for evaluation of TCR-Vβ repertoire in the bone marrow (BM) of these patients. We performed analysis of BM samples from healthy donors as a control group (n=8). Due to low amount of donor samples the maximal value each of the 24 subclones (for CD4+ (T-helpers - Th) and CD8+ cells (T-cytotoxic cells - TCL)) was accepted as threshold. We concluded the presence of clonal expansion if TCR subclone exceeded this threshold. We identified different T-cell subpopulations in all 3 groups of AA and healthy donors by flow cytometry: double positive T-cells (CD3+CD4+CD8+), double negative T-cells (CD3+CD4- CD8-), Th (CD3+CD4+), TCL (CD3+CD8+), NK-T-cells (CD3+CD56+) out of CD3+ cells. Among Th and TCL cells was determined naive T-cells (CD28+CD95-), effector T-cells (CD28-CD95+), memory T-cells (CD28+CD95+), regulatory T-cells (CD4+CD127-CD25high) and subpopulations Th and TCL co-expressed PD-1 and PD-L1. Multiple comparisons were assessed by ANOVA or Kruskal Wallis test by GraphPad Prism software.Results. In our study all 30 AA patients had an immunodominant TCR-Vβ clones among Th and/or TCL cells. We identified the most common clonotypes in comparison with healthy donors - Vβ1, Vβ2, Vβ3 among the Th cells and Vβ3, Vβ9, Vβ13.1 among the TCL cells. In ND group Vβ1 was highly expanded in 5 (38.5%), Vβ3 - in 7 (53.8%) pts among Th, and Vβ3 - in 3 (23.1%) and Vβ9 - in 4 (30.8%) out of 13 pts among TCL. In OR group Vβ2 expansion was in 4 (40%) and Vβ3 - in 5 (50%) pts among Th; Vβ3 in 6 (60%) and Vβ9 in 6 (60%) out of 10 pts among TCL. In NR group the most frequent was Vβ13.1 clone in TCL - in 3 (42.9%) out of 7 pts. In NR group in overall clonal expansion was less frequent than in ND and OR groups. We also analyzed the previously mentioned subpopulations of T-cells in patients with AA in three groups (ND, OR, NR) compared to healthy donors (table 1). We obtained significant differences in the count of naive Th and TCL cells, memory T-cells in all three groups of AA patients compared to donors: proportion of naive Th and TCL cells was significantly higher and proportion of memory Th cells was lower in the donor group than in AA pts. The percent of TCL effectors was higher in ND AA pts compare to donors. We also found that cell count of activated Th (CD4+CD25+) was higher in the group of refractory pts.In OR pts proportion of PD-1-positive Th was higher than in donors. In NR pts Th and TCL co-expressed with PD-L1 were lower compare to donors (table 1).Conclusions. In our study we found immunodominant clonotypes in different AA pts and depletion of the pool of naive T cells. Dynamic observation of changes in the most common clonotypes in AA pts during treatment will provide suitable therapy tactics (allogenic bone marrow transplantation or IST). [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Interaction of erythrocytes in the process of pair aggregation in blood samples from patients with arterial hypertension and healthy donors: measurements with laser tweezers

Two-channel laser tweezers are used to measure the interaction kinetics of two erythrocytes at the initial stage of aggregation, i.e., the formation of a pair aggregate in vitro. The study of erythrocytes interaction is important both for understanding the fundamental aggregation mechanisms and for evaluating the differences in kinetics and dynamics of aggregation, depending on the presence or absence of diseases that disturb the blood flow parameters and, therefore, the oxygen supply to tissues. We analyse the kinetics and dynamics of pair aggregation of erythrocytes in blood samples from more than 60 patients with arterial hypertension, as well as from a group of healthy donors. The results show that both kinetics and dynamics of erythrocyte aggregation are changed in the presence of the considered pathology.

PO-129 In vitro radiosensitivity and repair kinetics of PBMCs from prostate cancer patients and healthy donors evaluated by comet assay

IntroductionA high cellular radiosensitivity is connected with a risk for development of severe side effects after radiotherapy. In this study we have attempted to find a correlation between the initial radiosensitivity of in vitro irradiated peripheral blood mononuclear cells (PBMC) of prostate cancer patients and the adverse side effects of radiotherapy.Material and methodsPBMCs isolated from 7 prostate cancer patients, before the onset of radiotherapy and 3 healthy men were exposed in vitro to 2 Gy of x-ray radiation (Varian, 6 MeV). Following irradiation cell death was measured using the Annexin V/PI assay. DNA repair kinetics (0 and 24 hour after irradiation) was performed using the comet assay, and the results were expressed as% of tail DNA. Acute toxicity were graded according to the EORTC radiation morbidity scoring scale.Results and discussionsOur data show that the in vitro irradiation of PBMCs in prostate cancer patients initially caused a significantly higher DNA damage than in the control group of healthy donors. After a repair time of 24 hour, samples from all healthy donors showed no residual DNA damage (average 2.6%) in PBMCs. If all patients are observed for 0 hour and 24 hour after in vitro PBMCs irradiation then there is a significant reduction in the degree of DNA damage, from 35.5 to 30.4 tail% DNA. What is also important is that 4 patients had a very low level of DNA damage, which can be considered as normal cellular reaction to irradiation, while 3 patients had a very high level of damage. Also, in 5 out of 7 examined prostate cancer patients, there was an increase in the percentage of PBMCs in the early and late stages of apoptosis 24 hour after in vitro irradiation of their PBMCs. Most patients had mild side reactions to radiotherapy and were graded as grade 1 (per EORTC scale). Only in 1 of 7 patients observed side effects are classified as grade 2 and this is in correlation with a very high level of DNA damage (82.25% initial, versus 64.77% 24 hour after in vitro irradiation).ConclusionOur study was insufficient to reveal the relationship between the risk of developing side effects to radiotherapy and the sensitivity of PBMCs irradiated in vitro, measured by comet assay. A larger number of patients and further studies are necessary to confirm the potential application of the comet assay.

Generation of tolerogenic dendritic cells using Lactobacillus rhamnosus and Lactobacillus delbrueckii as tolerogenic probiotics.

Systemic lupus erythematosus (SLE) concurs with excessive uncontrolled inflammatory immune responses that lead to the loss of immune tolerance. Dendritic cells (DCs) are important and determinant immune cells that regulate immune responses. Tolerogenic DCs with regulatory markers and cytokines could induce regulatory immune cells and responses. Tolerogenic probiotics are capable of producing regulatory DCs from monocytes in in vitro conditions. The purpose of this study was to evaluate the effect of Lactobacillus delbrueckii and Lactobacillus rhamnosus on the production of DCs in an in vitro condition. Peripheral blood mononuclear cellswere isolated from the healthy and SLE donors. Monocytes were cultured with optimized concentrations of granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4) to produce immature DCs (IDCs). An IDC uptake assay was performed, and IDCs of healthy and SLE donors were divided into three subgroups following 48 hours of treatment with GM-CSF and IL-4, along with L. delbrueckii, L. rhamnosus, and mixed probiotics for the production of tolerogenic DCs. The surface expression of Human Leukocyte Antigen-antigen D Related (HLA-DR), CD86, CD80, CD83, CD1a, and CD14 was analyzed using flow cytometry, and the gene expression levels of indoleamine 2,3-dioxygenase (IDO), IL-10, and IL-12 were measured using real-timepolymerase chain reaction. We observed significantly reduced expression of costimulatory molecules and other surface markers in the probiotic-induced mature DCs (MDCs) in both healthy and SLE donor groups in comparison with lipopolysaccharide (LPS)-induced MDCs. In addition, the expression of IDO and IL-10 increased, whereas IL-12 decreased significantly in probiotic-induced MDCs compared with LPS-induced MDCs. IDCs and especially mature tolerogenic DC of SLE patients highly expressed IDO. The results of the current study suggested that live probiotics could modify properties of DCs to modulatory cells, which might contribute to the induction of tolerance and renovation of immune hemostasis.

ASSOCIATION OF THE POLYMORPHIC VARIANT RS1061622 OF THE TNFRSFB1 GENE WITH GENETIC PREDISPOSITION TO ESSENTIAL ARTERIAL HYPERTENSION AND CHANGES IN THE BLOOD LIPID PROFILE

The association of the TNFRSFB1 gene polymorphic variant rs1061622, which encodes the second type receptor (TNFRII) to TNFα with the development of essential arterial hypertension (EH, stages I–II) was investigated. Statistically significant differences in the frequencies of alleles and genotypes by this polymorphic marker were revealed for the group of healthy people and patients with EH. The risk of developing this disease was almost double in carriers of the allele G (OR = 1.904 (95DI: 1.337–2.709)). The effect of rs1061622 of the TNFRSFB1 gene on the lipid metabolism (total cholesterol, lowdensity lipoprotein cholesterol (LDL–C), high-density lipoprotein cholesterol (HDL–C), triglycerides), as well as the atherogenicity index in the group of healthy donors and EH patients, carriers of certain genotypes for this polymorphic marker of the TNFRSFB1 gene, was studied. An increase in the atherogenic lipid fraction (LDL–C) level in the plasma is associated with the presence of the allele G in the genotype. In healthy donors, either homozygous or heterozygous for the G allele, the content of HDL–C is significantly lower than in TT genotype carriers. The obtained results indicate that the polymorphic variant T>G rs1061622 of the TNFRSF1B gene is probably involved in the predisposition of people living in Karelia to EH (stages I–II). The pathogenetic effect of the TNFRSFB1 gene polymorphism on the formation of this disease is possibly accomplished through modulation of the levels of various lipid fractions.

Glycosylation of random IgG distinguishes seropositive and seronegative rheumatoid arthritis

The N-glycosylation of human immunoglobulins, especially IgGs, plays a critical role in determining affinity of IgGs towards their effector (pro- and anti-inflammatory) receptors. However, it is still not clear whether altered glycosylation is involved in only antibody-dependent disorders like seropositive rheumatoid arthritis (RA) or also in pathologies with similar clinical manifestations, but no specific autoantibodies like seronegative RA. The clarification of that uncertainty was the aim of the current study. Another study aim was the detection of specific glycan forms responsible for altered exposure of native glycoepitopes. We studied sera from seropositive RA (n = 15) and seronegative RA (n = 12) patients for exposure of glycans in native IgG molecules, followed by determination of specific glycans by capillary electrophoresis with laser-induced fluorescent detection (CE-LIF). Aged-matched groups of normal healthy donors (NHD) and samples of intravenous immunoglobulin IgG preparations (IVIG) served as controls. There was significantly stronger binding of Lens culinaris agglutinin (LCA) and Aleuria aurantia lectin (AAL) lectins towards IgG from seropositive RA compared to seronegative RA or NHD. CE-LIF analysis revealed statistically significant increases in bisecting glycans FA2BG2 (p = .006) and FABG2S1 (p = .005) seropositive RA, accompanied by decrease of bisecting monogalactosylated glycan FA2(6)G1 (p = .074) and non-bisecting monosialylated glycan FA2(3)G1S1 (p = .055). The results suggest that seropositive RA is distinct from seronegative RA in terms of IgG glycan moieties, attributable to specific immunoglobulin molecules present in seropositive disease. These glycans were determined to be bisecting GlcNAc-bearing forms FA2BG2 and FABG2S1, and their appearance increased the availability of LCA and AAL lectin-binding sites in native IgG glycoepitopes.

The tyrosine-kinase inhibitor sunitinib targets vascular endothelial (VE)-cadherin: a marker of response to antitumoural treatment in metastatic renal cell carcinoma

BackgroundVascular endothelial (VE)-cadherin is an endothelial cell-specific protein responsible for endothelium integrity. Its adhesive properties are regulated by post-translational processing, such as tyrosine phosphorylation at site Y685 in its cytoplasmic domain, and cleavage of its extracellular domain (sVE). In hormone-refractory metastatic breast cancer, we recently demonstrated that sVE levels correlate to poor survival. In the present study, we determine whether kidney cancer therapies had an effect on VE-cadherin structural modifications and their clinical interest to monitor patient outcome.MethodsThe effects of kidney cancer biotherapies were tested on an endothelial monolayer model mimicking the endothelium lining blood vessels and on a homotypic and heterotypic 3D cell model mimicking tumour growth. sVE was quantified by ELISA in renal cell carcinoma patients initiating sunitinib (48 patients) or bevacizumab (83 patients) in the first-line metastatic setting (SUVEGIL and TORAVA trials).ResultsHuman VE-cadherin is a direct target for sunitinib which inhibits its VEGF-induced phosphorylation and cleavage on endothelial monolayer and endothelial cell migration in the 3D model. The tumour cell environment modulates VE-cadherin functions through MMPs and VEGF. We demonstrate the presence of soluble VE-cadherin in the sera of mRCC patients (n = 131) which level at baseline, is higher than in a healthy donor group (n = 96). Analysis of sVE level after 4 weeks of treatment showed that a decrease in sVE level discriminates the responders vs. non-responders to sunitinib, but not bevacizumab.ConclusionsThese data highlight the interest for the sVE bioassay in future follow-up of cancer patients treated with targeted therapies such as tyrosine-kinase inhibitors.