Supernatant Of Blood Cells Research Articles

Cell activation, PD-1 expression and in vitro cytokine production in patients with juvenile systemic lupus erythematosus.

Juvenile systemic lupus erythematosus (jSLE) is known to be more severe and with a higher frequency of renal and central nervous system impairment when compared to systemic lupus erythematosus in adults. The study of immunological characteristics of jSLE patients might help to envisage better treatment strategies to reduce the burden of the disease. To characterize peripheral lymphocytes, assessing activation markers, and PD-1 expression on T cells; to evaluate in vitro cytokine expression upon stimulation in jSLE patients and age-matched controls. Eighteen jSLE patients on low disease activity and 25 matched healthy adolescents were evaluated for immune activation and PD-1 expression on peripheral blood lymphocytes by flow cytometry. Twenty-one cytokines were assessed by X-MAP technology after in vitro stimulation of peripheral blood with phytohemagglutinin. jSLE patients had lower numbers of CD4 T, CD8 T, B, and NK cells; higher central memory CD8 T cell percentages were noted in jSLE adolescents in comparison with controls (p = 0.014). B cells subsets showed a higher percentage of exhausted memory subset than controls (p = 0.014). The expression of PD-1 on CD4 T and CD8 T cells did not show relevant changes in jSLE adolescents. After stimulation of peripheral blood, cell supernatant of jSLE patients showed a trend to lower concentrations of IL-10 (p=0.080) and higher concentrations of IL-23 (p = 0.063) than controls. jSLE patients on low disease activity maintain lymphopenia of all subsets, with a B cell profile of exhaustion. Upon in vitro stimulation, peripheral blood cell supernatant showed a shift to IL-23, suggesting a role of inhibitors of this cytokine as another potential therapeutic target for those patients.

Red blood cell supernatant increases activation and agonist-induced reactivity of blood platelets

IntroductionTransfusion of “older” packed red blood cells (PRBCs) in patients with cardiovascular disorders (CVD) may be associated with an increased risk of pro-thrombotic events, but the underlying mechanisms are poorly understood. We hypothesized that the PRBC supernatant can activate blood platelets due to hemolysis-induced oxidative stress. MethodsEffects of the PRBC supernatants, and their filtrates (containing the soluble substances of molecular weight <10 kDa) prepared at day 1 and 42 of storage, from non-leukoreduced (D1 NLR, D42 NLR) and leukoreduced (D1 LR, D42 LR) PRBCs on PLT activation/reactivity and collagen-induced aggregation were measured by flow cytometry and turbidimetry, respectively. ResultsSupernatants display a stimulating effect on PLTs, which was manifested by a release of PLT-derived microparticles, generation of PLT aggregates, increased P-selectin expression on the membrane surface, and activation of integrin αIIbβ3. Moreover, supernatants interacted in a way that may be additive or synergistic with collagen or with ADP. The pre-storage LR did not affect the levels of PLT activation markers. The enhanced PLT activation was presumably mediated by free hemoglobin and/or the products of its breakdown, accumulating in the PRBC milieu, and their ability to trigger the ROS generation. Additionally, collagen-induced PLT aggregation was increased by low molecular weight substances possibly derived from the residual leukocytes and PLTs present in PRBCs. ConclusionTransfusion of aged PRBCs may result in the hyper-activity of PLTs, which, at least in part, could be a cause of transfusion-related thrombotic complications reported in CVD patients.

Proteins and immunohistochemical markers of breast diseases

In this work, we have compared malignant and non-malignant diseases of the mammary gland using 8 proteins: HRG, MUC1, PAI-1, HSP90αA1, CDH1, ERα, PGR and IL-12. Their concentrations in the supernatants of blood cells and breast biopsies were compared in terms of spontaneous production, induced by a polyclonal activator and after exposure to biopsy samples of the HLDF differentiation factor, as well as the indices of the effect of the polyclonal activator and HLDF on the protein production. In addition, the correlation relationships of the above indicators with the expression of markers of the epithelial-mesenchymal transition: collagen type II (CII), β-1 integrin (CD29) and cadherin-E (CDH1) were studied. The study revealed statistically significant differences in the concentration of HRG in the supernatant of blood cells, IL-12 during spontaneous production by biopsy specimens, PGR production of biopsy specimens induced by the polyclonal activator, CDH1 and IL-12 production biopsy specimens exposed to HLDF. According to the influence index of the polyclonal activator and HLDF, statistically significant differences were found for CDH1production. Comparison of non-specific invasive carcinoma biopsy specimens and non-malignant breast diseases by means of the markers of the epithelial-mesenchymal transition revealed statistically significant differences in CD29 expression and the lack of differences in the expression of CDH1 and CII. This indicates the presence of cell atypia in samples of non-malignant breast diseases; it is confirmed by the recognized correlation between the production of certain proteins and the expression of the epithelial-mesenchymal transition markers.

Personalized Approach to Determination of Histidine-Rich Glycoprotein and E-Cadherin in Supernatants of Immunocompetent Blood Cells and Breast Biopsy Specimens in Breast Malignant and Non-Malignant Disease.

The material of patients with invasive carcinoma of no special type (ICNT) and nonmalignant diseases (ND) of the mammary gland was studied. When comparing the concentrations of histidine-rich glycoprotein (HRG) and E-cadherin (CDH1), statistically significant differences between ICNT and ND by HRG in the supernatant of blood cells and its spontaneous production by biopsies and by CDH1 at its induced production, as well as by influence indices of polyclonal activators on the production of CDH1 were found. When comparing the expression of immunohistochemical markers, no statistically significant differences between ICNT and ND were obtained.

Aged Human Stored Red Blood Cell Supernatant Inhibits Macrophage Phagocytosis in an HMGB1 Dependent Manner After Trauma in a Murine Model.

Red blood cell transfusions in the setting of trauma are a double-edged sword, as it is a necessary component for life-sustaining treatment in massive hemorrhagic shock, but also associated with increased risk for nosocomial infections and immune suppression. The mechanisms surrounding this immune suppression are unclear. Using supernatant from human packed red blood cell (RBC), we demonstrate that clearance of Escherichia coli by macrophages is inhibited both in vitro and in vivo using a murine model of trauma and hemorrhagic shock. We further explore the mechanism of this inhibition by demonstrating that human-stored RBCs contain soluble high-mobility group box 1 protein (HMGB1) that increases throughout storage. HMGB1 derived from the supernatant of human-stored RBCs was shown to inhibit bacterial clearance, as neutralizing antibodies to HMGB1 restored the ability of macrophages to clear bacteria. These findings demonstrate that extracellular HMGB1 within stored RBCs could be one factor leading to immune suppression following transfusion in the trauma setting.

P64: Effect of polyclonal activators on cytokine-producing capacity of blood immunocompetent cells in adenoma and adenocarcinoma of the stomach

Adenoma and adenocarcinoma of the stomach are related to precancerous states and considered as successive stages of disregeneratory process proceeding against a background of chronic inflammation relying on cytokine-producing function of immune cells. Objective Evaluation of cytokine-producing function of blood immunocompetent cells under the influence on polyclonal activators in patients with adenoma and adenocarcinoma of the stomach. Materials and Methods Peripheral blood of 56 patients including 32 patients with adenomas and chronic atrophic gastritis and 24 patients with adenocarcinoma of the stomach was taken before surgery and biopsy. Peripheral blood of 30 healthy individuals was used as a control. To assess the status of the gastric mucosa, concentrations of pepsinogen I and pepsinogen II were determined in addition to instrumental methods. Cytokine-producing capacity of blood cells was determined using a polyclonal activator (PA) composed of PHA (4 μg/ml), conA (4 μg/ml) and LPS (2 μg/ml). Concentrations of the following cytokines: IL-1β, IL- 1Ra, TNFα, IL-2, IL-6, IL-8, IL-10, IL-17, IL-18, MCP-1, VEGF and IFNγ in the supernatant of blood cells were measured with enzyme-linked immunosorbent assay (ELISA). The index of polyclonal activators unfluence (IPAI) on cytokine production was calculated: IPAI = A/B, where A is cytokine production induced by PA, B-spontaneous cytokine production level. Results In patients with stomach adenomas, serum concentration of pepsinogen I and the pepsinogen I/pepsinogen II ratio were significantly lower compared with healthy individuals indicating the development of adenoma combined with gastric mucosa atrophy. Pepsinogen I concentration was in inverse correlation with the percentage of Ki-67 positive epithelial cells, which was also in inverse correlation with IPAI of IFNγ possessing antiproliferative action. In patients with stomach adenomas,the increased IPAI of receptor antagonist IL-1compared with the control was revealed, that could lead to inflammation incompleteness and its torpid course. In conjunction with IPAI, increase in IL-18 level stimulates proliferative processes and contributes to disregeneratory changes in the stomach epithelium. Direct correlation between the ability of blood cells to the produce IL-1Ra under the PA influence and the number of metastases in the regional lymph nodes as well as reverse correlation between IPAI of IL-2 and the number of low differentiated cells in the tumor were observed. In patient with stomach adenomas, degree of atrophy increases as the pepsinogen I/pepsinogen II ratio decreases, the ability of immune cells to produce IL-18 under the influence of PA stimulates proliferation of cells, including atypical. Conclusion Findings suggest the ability of blood cells to produce TNFα, IL-18 and IFNγ under the influence of polyclonal activators was higher in patients with stomach adenomas than in patients with stomach adenocarcinomas. In patients with adenocarcinomas the decline in the production of cytokines with both the proliferative and antiproliferative effects indicates radical changes in the cytokine-producing capability of immunocompetent cells when malignant neoplasm became mature.

Biochemical assessment of red blood cells during storage by (1)H nuclear magnetic resonance spectroscopy. Identification of a biomarker of their level of protection against oxidative stress.

Blood transfusion is an established therapeutic practice. The characteristics of blood components at different storage times are expected to affect the efficacy of transfusion therapy. Metabolic profiling by nuclear magnetic resonance (NMR) spectroscopy requires little or no sample treatment and allows identification of more than 50 soluble metabolites in a single experiment. The aim of this study was to assess the metabolic behaviour of red blood cells during 42 days of storage in blood bank conditions. Red blood cells (RBC), collected from eight healthy male donors, aged 25-50 years, were prepared as prestorage leukoreduced erythrocyte concentrates and stored under standard blood bank conditions. Samples taken at various storage times were separated in two fractions: the supernatant, recovered after centrifugation, and the red blood cell lysate obtained after protein depletion by ultrafiltration. The metabolic profile of the red blood cells was determined from analysis of (1)H-NMR spectra. The red blood cell supernatant was studied to track the consumption of the preservative additives and to detect and quantify up to 30 metabolites excreted by the erythrocytes. The NMR spectra of the RBC lysate provided complementary information on some biochemical pathways and set the basis for building a time-dependent red blood cell metabolic profile. We proved the analytical power of (1)H-NMR spectroscopy to study red blood cell metabolism under blood bank conditions. A potential biomarker able to provide information on the level of cellular oxidative stress protection was identified. Our data support the hypothesis that a more detailed knowledge of metabolic modifications during storage opens the way to the development of new and more effective protocols for red blood cell conservation and patient-oriented transfusion therapy.

Measurement of ESAT6-induced IFNγ responses adjunct with CXCL9 increases the rate of diagnosis of active tuberculosis in an endemic region

Due to difficulties in direct diagnosis of Mycobacterium tuberculosis infection where site-specific specimens are not available, indirect methods of testing for infection are required. M. tuberculosis early secreted antigen target-6 (ESAT6) induced IFN-γ responses are specific, but do not differentiate between latent and active TB. The use of adjunct biomarkers for TB diagnosis has been proposed, such as the chemokines: CXCL9, CXCL10 and CCL2.ESAT6-induced IFN-γ CXCL9, CXCL10 and CCL2 was measured in whole blood cell supernatants of patients with pulmonary tuberculosis (PTB, n=36) and extrapulmonary TB (ETB, n=31) and compared with healthy endemic controls (EC, n=33).ESAT6-induced IFN-γ responses were positive in 32% of TB cases as compared with 15% of EC cases (p=0.048). ESAT6-induced CXCL9 responses were positive in 42% of TB cases and 15% of EC cases (p=0.006). ESAT6-induced-CXCL10 and -CCL2 responses did not discriminate between TB and EC groups. Measurement of IFN-γ or CXCL9 together diagnosed TB (53%) cases and was significant as compared with EC (p=0.014) cases. IFN-γ and CXCL10 together did not increase the number of TB cases diagnosed.Within TB groups, ESAT6-IFN-γ/CXCL9-based detection increased to 53% in PTB (p=0.031) and 54% in ETB (p=0.021), with comparable diagnosis in less severe extrapulmonary TB (L-ETB, 55%) and severe disseminated extrapulmonary TB (D-ETB, 50%). Given that 47% of TB cases remained undetected, this study shown that ESAT6-induced IFNγ and CXCL9 can support diagnosis, but must be supported by clinical correlation and other relevant investigations.

Red Blood Cell Supernatant Potentiates LPS-Induced Proinflammatory Cytokine Response From Peripheral Blood Mononuclear Cells

Allogeneic blood transfusion has an immunomodulatory capacity on its recipients through accumulation of immunologically active substances with blood storage, and prestorage leukoreduction reduces many of these mediators. We investigated lipopolysaccharide (LPS)-induced cytokine response of peripheral blood mononuclear cells (PBMCs) exposed to packed red blood cell (PRBC) supernatants from leukoreduced (LR) or non-leukoreduced (NLR) units with variable duration of storage. PRBC units were collected with or without leukoreduction on Day 0 before routine storage. The plasma fraction (supernatant) was isolated from LR and NLR units after 1 day (D1) or 42 days (D42) of storage and exposed to PBMCs versus control media for 24 h, then with LPS for an additional 24 h. Cell supernatants were analyzed for IL-1beta, IL-6, IL-8, IL-10, and TNF-alpha by cytokine bead array. IL-1beta, TNF-alpha, and IL-6 were significantly elevated in PRBC groups versus control. D42 NLR PRBC supernatant significantly increased secretion of IL-1beta and IL-6 compared to D1 NLR PRBC supernatant. LR significantly attenuated the cytokine response of IL-1beta. Thus, PRBC supernatant potentiates proinflammatory LPS-induced cytokine secretion from PBMCs. This response is accentuated with storage duration and partially attenuated with leukoreduction. These findings may partially explain the immune activation seen clinically after blood transfusion.

Red blood cell supernatant induces regulatory t cells independent of leukoreduction or free hemoglobin

Baumgartne, Joel M. MD; Moore, Ernest E. MD, FACS; Banerjee, Anirban PhD; Silliman, Christopher MD, PhD; McCarter, Martin MD Author Information

Innate immune responses in Lyme borreliosis: enhanced tumour necrosis factor-α and interleukin-12 in asymptomatic individuals in response to live spirochetes

Innate immunity is important for early defence against borrelia spirochetes and should play a role in the clinical outcome of the infection. In order to study early cytokine responses, in vitro differentiated dendritic cells (DCs) and whole blood cells from 21 patients with different clinical outcomes of Lyme neuroborreliosis were stimulated with live borrelia spirochetes. The borrelia-induced secretion of interleukin (IL)-4, IL-10, IL-12p70, interferon (IFN)-gamma and tumour necrosis factor (TNF)-alpha in DCs and IL-1beta, IL-6, IL-8, IL-10, IL-12p70, TNF-alpha, regulated upon activation normal T cell expressed and secreted (RANTES), monocyte chemoattractant protein (MCP)-1, macrophage inflammatory protein (MIP)-1alpha, MIP-1beta and eotaxin in whole blood cells was measured by enzyme-linked immunospot (ELISPOT) and multiplex arrays, respectively. We found increased numbers of TNF-alpha-secreting DCs (P = 0.018) in asymptomatic seropositive individuals compared to patients with subacute neuroborreliosis and seronegative controls. Asymptomatic individuals were also found to have elevated levels of IL-12p70 (P = 0.031) in whole blood cell supernatants compared to seronegative controls. These results are in line with previous experiments using cells of the adaptive immune response, indicating that strong T helper type 1 (Th1) proinflammatory responses might be associated with a successful resolution of Lyme disease.

Inter- and intraindividual variation of endotoxin- and beta(1 --> 3)-glucan-induced cytokine responses in a whole blood assay.

Inflammatory airway responses to bioaerosols and to their active compounds, such as endotoxin and beta(1 --> 3)-glucan, vary between individuals. These differences may be explained by variation in cytokine responsiveness, which can be assessed by in vitro stimulation tests with isolated blood leukocytes or lung macrophages. In large-scale population studies, ex vivo induced cytokine production may also be tested with a more simple 'whole blood assay' (WBA). However, applicability of a WBA to characterize a subject's responsiveness depends largely on its reproducibility. This study was conducted to: 1) assess the within- and between-subject variability in cytokine production in a WBA after stimulation with endotoxin or beta(1 --> 3)-glucan; and 2) to determine under which conditions this test is most discriminating between subjects and most reproducible within subjects. Blood was collected from 14 healthy volunteers, of whom 10 also participated on a second occasion. Each blood sample was used in two WBA tests; the first WBA was initiated two hours and the second 26 hours after venapuncture. The WBA test itself comprised overnight incubation with serial dilutions of endotoxin [lipopolysaccharide (LPS)] and curdlan (a beta(1 --> 3)-glucan), after which blood cell supernatant was collected. Interleukin(IL)-1beta, IL6, IL8 and tumor necrosis factor alpha (TNFalpha) were determined in the supernatant. In all individuals, a dose-dependent production of cytokines was observed for both LPS and curdlan. For all cytokines, variation between subjects was higher than within subjects, and this was most pronounced for IL1beta and IL6. There was moderate-to-high correlation in the induced release of all four cytokines, and between cytokine release induced by LPS or curdlan. Optimal stimulation concentrations were 6.25 and 12.5 ng/mL for endotoxin and 12500 and 25000 ng/mL for curdlan. Cytokine production in WBA initiated 26 hours after venapuncture showed lower between-subject and larger within-subject variance, thus favoring an early initiation of the assay. In conclusion, measuring endotoxin- or glucan-induced cytokine production in a WBA initiated within two hours after venapuncture appears to be an effective method to determine a person's cytokine responsiveness, at least in healthy naive subjects.

Detection of a gliotoxic activity in the cerebrospinal fluid from multiple sclerosis patients

We recently showed that peripheral blood cell supernatants from multiple sclerosis (MS) patients, containing reverse transcriptase activity and retroviral RNA from the newly human identified multiple sclerosis retrovirus (MSRV), also secrete a cytotoxin which induces death of primary mouse cortical glial cells. We have hypothesized that macrophages could release this cytotoxin in the cerebrospinal fluid. The cerebrospinal fluid cytotoxicity from 166 patients with various neurological diseases (including MS patients) was tested on glial cells in vitro. Our bioassay shows that a glial cytotoxic activity is significantly present in cerebrospinal fluid from patients with relapsing-remitting MS at relapse. Since this cytotoxic activity seems to correlate with active cases of MS, it may represent a critical pathogenic factor in the neuropathology of MS.

IgA content of frozen-thawed-washed red blood cells and blood products measured by radioimmunoassay.

The IgA concentration of various blood products was examined by a radioimmunoassay, and the lowest IgA concentrations were seen in frozen-thawed-washed red blood cells. The routine Regional Blood Transfusion Centre preparation contained a mean IgA concentration of 0.117 mg/l (n=21) in the frozen-thawed-washed red blood cell supernatant fluid. Further washing of frozen-thawed-washed red blood cells with saline reduced supernatant IgA concentrations to below the limit of IgA detection (less than 0.003 mg/l). Ordinary washed red blood cells and other blood plasma fractions contained substantial quantitites of IgA, supporting the view that frozen-thawed-washed red blood cells should be used for transfusion of patients at risk of developing reactions to IgA, and the need for caution in the use of other blood products in such patients.

Photo-Hemolysis Associated with Protoporphyrinemia: Preliminary Report

Since 1961 when Magnus, Jarrett, Prankard, and Rimington (1) first described a new disease entity, viz. erythropoietic protoporphyria, there have been reports of similar cases by several investigators (2, 3, 4, 5, 6). In all the reported cases, there were markedly elevated erythrocyte protoporphyrin levels associated with cutaneous photosensitivity. This form of photosensitivity may express itself in diverse cutaneous lesions including erythema, vesicles, wheals and eczema. We wish to report the preliminary results of studies concerning the in vitro hemolysis of red blood cells by ultraviolet light using cells from a patient having erythropoietic protoporphyrinemia. The patient is a seven year old girl who has vesicular and crusted skin lesions on the light exposed areas on and off since the age of 4 and who has been under our observation for the past 2 years. Erythrocyte protoporphyrin blood levels (7) were assayed on 4 occasions and were found to be significantly elevated. Preparation of Red Blood Cells for Hemolysis Studies: In each test run, the effects of ultraviolet light on the patient's red blood cells and on the red blood cells of a normal control subject were compared. Prior to irradiation, the cells were washed three times and resuspended in a 1:400 dilution of a 0.9% NaC1 phosphate buffer. Light Source: The red blood cells were irradiated with light from a bank of 4 Westinghouse F20 T12 BL blacklight fluorescent tubes. The emission spectrum of these tubes is mainly between 3200A° and 4500A°. In order to minimize the infrared radiation and the minute amount of radiation below 3200A the light was directed through a Corning 1-69 filter and through a window glass filter, 3 mm thick. The red blood cells were enclosed in quartz receptacles, and irradi* From the Department of Dermatology of New York University Schools of Medicine, New York, New York. This study was supported by Grant Number DA-49-193-MD-2275 from the U.S. Army Medical Research and Development Command. Technical Assistance given by Mary E. Moragne, B.S. Received for publication October 30, 1963. ated at a target distance of 6.25 centimeters. The intensity of the incident radiation was approximately 1100 microwatts per centimeter square. Measurement of Hemolytis was carried out by two methods: 1) direct cell counts: aliquots of the red blood cell suspensions were counted in a No. 500 Levy Counting Chamber (hemocytometer), at 10 minute intervals. Hemolysis was evident in the reduction of the number of red cells counted; 2) spectrophotometrically: the concentration of hemoglobin in the red blood cell supernatant was measured in a Coleman junior spectrophotometer.