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Background Asthma is a chronic respiratory disease with marked clinical and pathophysiological heterogeneity. Specific pathways are thought to be involved in the pathomechanisms of different inflammatory phenotypes of asthma; however, direct in vivo comparison has not been performed. Methods We developed mouse models representing three different phenotypes of allergic airway inflammation-eosinophilic, mixed, and neutrophilic asthma via different methods of house dust mite sensitization and challenge. Transcriptomic analysis of the lungs, followed by the RT-PCR, western blot, and confocal microscopy, was performed. Primary human bronchial epithelial cells cultured in air-liquid interface were used to study the mechanisms revealed in the in vivo models. Results By whole-genome transcriptome profiling of the lung, we found that airway tight junction (TJ), mucin, and inflammasome-related genes are differentially expressed in these distinct phenotypes. Further analysis of proteins from these families revealed that Zo-1 and Cldn18 were downregulated in all phenotypes, while increased Cldn4 expression was characteristic for neutrophilic airway inflammation. Mucins Clca1 (Gob5) and Muc5ac were upregulated in eosinophilic and even more in neutrophilic phenotype. Increased expression of inflammasome-related molecules such as Nlrp3, Nlrc4, Casp-1, and IL-1β was characteristic for neutrophilic asthma. In addition, we showed that inflammasome/Th17/neutrophilic axis cytokine-IL-1β-may transiently impair epithelial barrier function, while IL-1β and IL-17 increase mucin expressions in primary human bronchial epithelial cells. Conclusion Our findings suggest that differential expression of TJ, mucin, and inflammasome-related molecules in distinct inflammatory phenotypes of asthma may be linked to pathophysiology and might reflect the differences observed in the clinic.

// Ahmed Abdelbaset-Ismail 1 , Sylwia Borkowska 2 , Anna Janowska-Wieczorek 3 , Torsten Tonn 4 , Cesar Rodriguez 1 , Marcin Moniuszko 5 , Lukasz Bolkun 7 , Janusz Koloczko 7 , Andrzej Eljaszewicz 5 , Janina Ratajczak 1 , Mariusz Z. Ratajczak 1, 6 , Magda Kucia 1, 6 1 Stem Cell Institute at James Graham Brown Cancer Center, University of Louisville, KY, USA 2 Pomeranian Medical University, Szczecin, Poland 3 Department of Hematology, University of Alberta, Edmonton, Canada 4 Transfusion Medicine, Medical Faculty Carl Gustav Carus - Technische Universtitat Dresden, German Red Cross Blood Donation Service North East, Dresden, Germany 5 Department of Regenerative Medicine and Immune Regulation, Medical University of Bialystok, Bialystok, Poland 6 Department of Regenerative Medicine Medical University of Warsaw, Warsaw, Poland 7 Department of Hematology, Medical University of Bialystok, Bialystok, Poland Correspondence to: Mariusz Ratajczak, e-mail: mzrata01@louisville.edu Magda Kucia, e-mail: mjkuci01@louisville.edu Keywords: leukemia, lymphoma, germ line, FSH, LH Received: August 25, 2015 Accepted: November 21, 2015 Published: December 20, 2015 ABSTRACT We recently reported that normal hematopoietic stem cells express functional pituitary sex hormone (SexH) receptors. Here we report for the first time that pituitary-secreted gonadotrophins stimulate migration, adhesion, and proliferation of several human myeloid and lymphoid leukemia cell lines. Similar effects were observed after stimulation of human leukemic cell lines by gonadal SexHs. This effect seems to be direct, as the SexH receptors expressed by leukemic cells responded to stimulation by phosphorylation of MAPK p42/44 and AKT ser473 . Furthermore, in parallel studies we confirmed that human primary patient-derived AML and CML blasts also express several functional SexH receptors. These results shed more light on the potential role of SexHs in leukemogenesis and, in addition, provide further evidence suggesting a developmental link between hematopoiesis and the germline.

Targeted therapy of non-small cell lung cancer (NSCLC) demands a more accurate tumor classification that is crucial for patient selection in personalized treatment. MicroRNAs constitute a promising class of biomarkers and a helpful tool for the distinction between lung adenocarcinoma (AC) and squamous cell lung carcinoma (SCC). The aim of this study was to evaluate the impact of two different normalization strategies, using U6 snRNA and hsa-miR-103 as reference genes, on hsa-miR-205 and hsa-miR-21 expression levels, in terms of the classification of subtypes of NSCLC. By means of a quantitative real-time polymerase chain reaction (qRT-PCR) microRNA expression levels were evaluated in a classification set of 98 surgically resected NSCLC fresh-frozen samples, and validated findings in an independent set of 42 NSCLC samples. The microRNA expression levels were exploited to develop a diagnostic test using two data normalization strategies. The performance of microRNA profiling in different normalization methods was compared. We revealed the microRNA-based qRT-PCR tests to be appropriate measures for distinguishing between AC and SCC (the concordance of histologic diagnoses and molecular methods greater than 88%). Performance evaluation of microRNA tests, based on the two normalization strategies, showed that the procedure using hsa-miR-103 as reference target has a slight advantage (sensitivity 83.33 and 100% in classification and validation set, respectively) compared to U6 snRNA. Molecular tests based on microRNA expression allow a reliable classification of subtypes for NSCLC and can constitute a useful diagnostic strategy in patient selection for targeted therapy.

The inflammation underlying both atherosclerosis and acute coronary syndromes is strongly related to monocyte-related actions. However, different monocyte subsets can play differential roles in the formation and destabilization of atherosclerotic plaque as well as healing of damaged myocardial tissue. Monocytes are currently being divided into three functionally distinct subsets with different levels of CD14 (cluster of differentiation 14) and CD16 expression. Thus, there are classical CD14++CD16-, intermediate CD14++CD16+ and non-classical CD14+CD16++ monocytes. Here, we summarize the current knowledge on complex activities of different monocyte subsets in atherosclerosis and acute coronary syndromes. Moreover, we discuss which monocyte subsets can serve either as predictive biomarkers of cardiovascular risk or as potential targets used in atherosclerosis and its complications.

It is believed that the recently discovered interleukin 17-producing Th17 cells play a role in the pathogenesis of chronic inflammation in the course of obesity and diabetes.The purpose of our study was to complete data on this subject in children.We assessed Th17 cell levels in the peripheral blood of children diagnosed with central obesity (n = 14) and compared the results with data obtained in patients with newly diagnosed (n = 11) and long-term type 1 diabetes mellitus (n = 18), and in a control group as well (n = 24).(i) Children with central obesity were characterized by higher percentages of Th17 cells as compared to children from the control group; (ii) in the peripheral blood of patients with long-term type 1 diabetes the Th17 cell counts were higher compared to the control group; (iii) total plasma cholesterol concentration correlated positively with Th17/Treg cells ratio; and (iv) among patients with long-term diabetes, disease duration correlated positively with Th17 cell count and Th17/Th1 cell ratio.The results of our study indicate that Th17 cells may be involved in chronic inflammation accompanying obesity and type 1 diabetes mellitus in children.

Altered activities of ligands belonging to tumour necrosis factor (TNF) superfamily, namely B-cell activating factor (BAFF), a proliferation-inducing ligand (APRIL) and apoptosis inducing ligand (TRAIL) were demonstrated in several haematological diseases including acute lymphoblastic leukaemia (ALL). BAFF, APRIL and TRAIL provide crucial survival signals to immature, naive and activated B cells. These ligands are capable of activating a broad spectrum of intracellular signalling cascades that can either induce apoptosis or protect from programmed cell death. BAFF and APRIL, which can directly activate the NF-κB pathway, have been identified as crucial survival factors for ALL cells. Here, we have analyzed serum BAFF, APRIL and TRAIL concentrations in 48 patients with newly diagnosed ALL and 44 healthy volunteers. The levels of APRIL and BAFF were significantly higher in ALL patients as compared to healthy volunteers. In contrast, concentrations of TRAIL were significantly lower in ALL patients. Moreover, following induction, the levels of APRIL, but not BAFF or TRAIL, were significantly lower in a group of patients with complete remission (CR) as compared to non-respondent (NR) ALL patients. Furthermore, we demonstrated statistically significant differences in concentrations of APRIL between CR MRD-negative and CR, MRD-positive ALL patients. Notably detection of higher concentrations of APRIL was associated with shorter leukaemia-free survival and overall survival. Altogether, our data indicate that APRIL can play an important role in the pathogenesis of ALL and the measurement of APRIL levels can improve prognostication in ALL patients.

Background: Previously, we demonstrated that glucocorticoid (GC) treatment of asthmatic patients resulted in decreasing frequencies of monocyte subsets expressing CD16 and capable of releasing TNF-α. Here, we wished to analyze whether the active form of vitamin D, i.e. vitamin D3, referred to as 1α,25-dihydroxyvitamin D3 [1,25-(OH)2D3] can exert GC-like proapoptotic effects on CD16-positive monocytes and thus decrease the proinflammatory potential of these cells. Finally, we set out to investigate whether the addition of 1,25-(OH)2D3 would facilitate the use of lower doses of GC without decreasing their anti-inflammatory properties. Methods: Peripheral blood mononuclear cells collected from healthy individuals and asthmatic patients were cultured with 1,25-(OH)2D3 and/or varying doses of GC in the presence or absence of caspase inhibition. The cells were either directly stained for extracellular markers or prestimulated with lipopolysaccharide for the assessment of intracellular cytokine production and then analyzed by flow cytometry. Results: We found that 1,25-(OH)2D3 alone (and in combination with GC) decreased the frequency of CD14++CD16+ and CD14+CD16++ monocytes from asthmatic patients and significantly diminished TNF-α production by the monocytes. With regard to the CD14+CD16++ subset, the monocyte-depleting effects of 1,25-(OH)2D3 were abrogated in the presence of pan-caspase inhibitor, suggesting a proapoptotic mechanism of 1,25-(OH)2D3 action. Interestingly, we found that a combined treatment of 1,25-(OH)2D3 and GC allowed for a 5-fold reduction of the GC dose while maintaining their anti-inflammatory effects. Conclusions: This study has revealed novel immunomodulatory properties of 1,25-(OH)2D3 directed against monocyte subsets capable of TNF-α production. In addition, our data suggest that the introduction of 1,25-(OH)2D3 to anti-inflammatory therapy would possibly allow for the use of lower doses of GC.

Bisphenol A (BPA) is a phenolic compound being constituent of numerous everyday-used products. Additionally, good absorption through skin and gastrointestinal tract is an unquestionable evidence for high risk of BPA exposure and its possible influence on health. Effects of BPA action on cells and tissues are associated with its structural and functional similarities to steroid hormones. Here we investigated whether BPA could possibly influence immune cells through steroid receptors present on majority of these cells. In in vitro experiments with 200 nM and 1000 nM BPA concentrations we found that high levels affect activation of lymphocytes through increased CD25 expression, with no changes in functional response based on IFN-gamma production. We demonstrated that BPA influence not only phenotype of monocytes with increased frequency of CD14++CD16- subtypes, but also activation inhibition with decline of HLADR expression within monocytes. Similarly to lymphocytes, no changes were observed in context of monocytes function in BPA-exposed cells. Our study revealed that BPA actions are also associated with its direct role in modulation of immune system cells. We presume that further experiments would allow establishing connection between presented data and increased risk of cancer and metabolic diseases in subject exposed to bisphenol A.

Tumour necrosis factor-alfa (TNF-α) is an inflammatory cytokine with a wide spectrum of biological activity, including angiogenesis. Tumour necrosis factor-related apoptosis inducing ligand (TRAIL), which belongs to the TNF family of proteins, plays a role in the regulation of vascular responses, but its effect on the formation of new blood vessels (angiogenesis) is unclear. We analysed TRAIL concentrations in parallel with pro-angiogenic cytokines in serum and their expression in trephine biopsy (TB) in 56 patients with newly diagnosed IgG MM and 24 healthy volunteers. The study showed statistically higher concentrations of TRAIL and TNF-α, as well as of VEGF and its receptor, in MM patients compared to healthy volunteers and patients in advanced stages of the disease. Furthermore, we observed a significant decrease in all studied pro-angiogenic cytokines and significant increase of TRAIL concentration after anti-angiogenic therapy, with meaningful differences between responders (at least partial remission) and patients with progression during the induction treatment. It was also established that TRAIL correlated statistically and negatively with pro-angiogenic cytokines such as VEGF with its receptor and expression of VEGF and syndecan-1 in TB. In summary, our data indicate that in MM patients, both clinical course and treatment responsiveness are associated with dynamic yet corresponding changes of levels of TRAIL parallel pro-angiogenic mediators such as VEGF with its receptor and expression of VEGF and syndecan-1 in TB. Copyright © 2014 John Wiley & Sons, Ltd.

Graves' orbitopathy (GO) is characterized by orbital T cell infiltration. We evaluated the regulatory T (Treg) cell fractions induced with IGF-1 in Graves' disease (GD) with and without GO. Peripheral blood mononuclear cells (PBMCs) were obtained from 13 patients with GD without eye manifestations; 10 patients with active GO; and 12 patients with nodular goiter (NG). All the patients from GD, GO, and NG were subclinical hyperthyroid. We analyzed the expression of Treg cell markers (CD4, CD25, CD127-, Foxp3) on T cells and their ability to respond to IGF-1 stimulation. In patients with GD without GO, we found lowered percentages of CD4+ Foxp3+ cells, as compared to nodular goiter 1.77 vs. 5.42% (p=0.0276). Similarly, significantly reduced frequencies of CD4+CD25+CD127-Foxp3+ and CD4+CD25+CD127- cells were observed in GD patients as compared to nodular goiter patients with hyperthyreosis, (0.7 vs. 1.48%) (p=0.0071) and (14.5 vs. 37.2%) (p=0.0051), respectively. In GO with active GO, only the percentage of CD4+CD25+CD127- cells was found to be decreased versus nodular goiter (9.35 vs. 37.2) (p=0.0275). Stimulation of PBMC derived from GO patients with IGF-1 resulted in significant increase of frequency of both CD4+ Foxp3+ and CD4+CD25+CD127- Foxp3 cells. Decreased frequencies of peripheral blood CD4+CD25+CD127-Foxp3+ in patients with GD and GO could be an useful marker of autoimmune process and perhaps a possible target for future therapies. This is the first study demonstrating Treg-enhancing effects of IGF-1. Thus IGF-1 can be accounted for modulating Treg cell-related action in GO.

Over the past few years, there have been considerable advances in the treatments available to patients with metastatic or locally advanced NSCLC, particularly those who have progressed during first-line treatment. Some of the treatment options available to patients are discussed here, with a focus on checkpoint inhibitor immunotherapies (nivolumab and pembrolizumab) and antiangiogenic agents (bevacizumab, ramucirumab, and nintedanib). It is hypothesized that combining immunotherapy with antiangiogenic treatment may have a synergistic effect and enhance the efficacy of both treatments. In this review, we explore the theory and potential of this novel treatment option for patients with advanced NSCLC. We discuss the growing body of evidence that proangiogenic factors can modulate the immune response (both by reducing T-cell infiltration into the tumor microenvironment and through systemic effects on immune-regulatory cell function), and we examine the preclinical evidence for combining these treatments. Potential challenges are also considered, and we review the preliminary evidence of clinical efficacy and safety with this novel combination in a variety of solid tumor types. (C) 2016 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

Over the past few years, there have been considerable advances in the treatments available to patients with metastatic or locally advanced NSCLC, particularly those who have progressed during first-line treatment. Some of the treatment options available to patients are discussed here, with a focus on checkpoint inhibitor immunotherapies (nivolumab and pembrolizumab) and antiangiogenic agents (bevacizumab, ramucirumab, and nintedanib). It is hypothesized that combining immunotherapy with antiangiogenic treatment may have a synergistic effect and enhance the efficacy of both treatments. In this review, we explore the theory and potential of this novel treatment option for patients with advanced NSCLC. We discuss the growing body of evidence that proangiogenic factors can modulate the immune response (both by reducing T-cell infiltration into the tumor microenvironment and through systemic effects on immune-regulatory cell function), and we examine the preclinical evidence for combining these treatments. Potential challenges are also considered, and we review the preliminary evidence of clinical efficacy and safety with this novel combination in a variety of solid tumor types. (C) 2016 International Association for the Study of Lung Cancer. Published by Elsevier Inc. All rights reserved.

The essence of autoimmune thyroid disease (AITD) is loss of tolerance of own tissues caused by malfunction of T lymphocytes, which affects the production of antibodies reacting with particular cell structures and tissues. Foxp3(+) regulatory T cells (Tregs) take part in the regulation of immune response and play a leading role in developing immune tolerance through active suppression. The aim of the study was to estimate the expression of CD4+CD25(high), CD4+CD25+CD127(low)FoxP3(+) and CD4+ FoxP3 T cells in patients with Graves' disease (GD) (n = 24, median age 15.5 years), in patients with Hashimoto's thyroiditis (HT) (n = 30, median age 15 years) in comparison with sex- and age-matched healthy control subjects (n = 30, median age 15 years). Polychromatic flow cytometry using a FACSCalibur (BD Biosciences) cytometer was applied to delineate T regulatory cell populations. In untreated patients with Graves' disease and HT we observed a significant decrease in CD4+FoxP3 (p < 0.001, p < 0.01) and CD4+CD25(high) (p < 0.016, p < 0.048) T lymphocytes as compared to the healthy control subjects. After 6-12 months of L-thyroxine therapy in HT cases these phenotypes of Tregs were normalized, yet no such changes were observed during GD therapy. The analysis of CD4+CD25+CD127(low)FoxP3+ T cells in the peripheral blood revealed comparable percentages of these cells in patients with thyroid autoimmune diseases to the controls. We conclude that the reduction number of Tregs with CD4+CD25(high) and CD4+FoxP3 phenotype suggests their role in initiation and development of autoimmune process in thyroid disorders.

Despite widely acknowledged contributions of innate and adaptive immune systems to the pathogenesis of allergic diseases, mutual interactions occurring in vivo between components of those two systems have not been studied in sufficient detail. Here, we wished to investigate whether phenotypic features of monocytes and CD4+ T cells in allergic patients are reciprocally related. Therefore, we recruited 50 untreated house dust mite-sensitive allergic rhinitis patients and 29 non-atopic healthy individuals and performed comprehensive simultaneous flow cytometric analysis of mutual correlations between levels of CD14, CD16, CD163, CD206, CD124 (IL-4R), CD210 (IL-10R) and CD25, CD124, CD127 (IL-7R), CD210, ICOS expression on monocytes and CD4+ T cells, respectively. We found that CD163 monocyte expression in allergic but not healthy subjects is positively correlated with monocyte IL-10R, and, to a lesser extent, CD206, but not IL-4R expression. Levels of CD163 expression were not related to frequencies of CD14++CD16–, CD14++CD16+, and CD14+CD16++ monocyte subsets. In contrast to healthy controls, intensities of monocyte IL-10R in allergic individuals were significantly correlated with monocyte CD206 and IL-4R expression. In addition, levels of monocyte IL-4R and IL-10R monocyte expression were positively correlated to expression of IL-4R and IL-10R on CD4+ T cells in both groups of studied subjects. Interestingly, we demonstrated a significant positive correlation between levels of monocyte CD206 expression and levels of IL-10R and IL-4R expression on CD4+ T cells in allergic but not healthy individuals. In summary, we conclude that allergic rhinitis is associated with a number of phenotypic alterations of circulating monocytes and CD4+ T cells.

Until now, altered balance of Th1 and Th2 immune cells has been postulated to play an important role in the pathogenesis of autoimmune thyroid diseases (AITD). However, recent studies on thyroid diseases have suggested a new role for Th17 cells that have been classified as a new lineage, distinct from Th1, Th2 and Treg cells. Despite wide interest, the role of Th17 cells in the pathogenesis of inflammatory and autoimmune diseases is still debated. The aim of the study was to estimate the proportions of Th17/Treg T cells in peripheral blood from patients with Graves' disease (GD; n = 29, mean age 15.4 ± 5.1 years), Hashimoto's thyroiditis (HT; n = 39, mean age 15.2 ± 4.1 years) and in healthy controls (n = 49, mean age 14.8 ± 3 years). Polychromatic flow cytometry and several fluorochrome-conjugated monoclonal antibodies were applied to delineate Th17 and Treg cells. The analysis of Th17/Treg T cell proportions in peripheral blood from patients with Graves' disease revealed significantly lower ratios of CD4 + IL17+/CD4 + CD25 + CD127 - (p 0.0021) and CD4 + IL17+/CD4 + CD25 + CD127 - FoxP3 + (p 0.0031) than in the control group. In addition, in the case of HT, we observed a significant decrease in the ratios of CD4 + IL17+/CD4 + CD25 + CD127 - (p 0.0001) and CD4 + IL17+/CD4 + CD25 + CD127 - FoxP3 + (p 0.0001) T cells in comparison to healthy children. In patients with untreated GD, a statistically significant positive correlation was found between the proportions of CD4 + IL17+/CD4 + CD25 + CD127-, CD4 + IL17+/CD4 + CD25 + CD127 - FoxP3+ T cells and the TRAbs (R = 0.71, p 0.029; R = 0.72, p 0.026, respectively) and a positive correlation was noted between the percentage of CD4 + CD - IL - 17 + T cells and the level of TSAbs (R = 0.66, p 0.037). We conclude that the changes in the proportion of Th17/Treg T cells in peripheral blood and their significant relationship with the level of anti-thyroid antibodies indicate an involvement of these cells in the pathogenesis of AITD.

B-cell activation factor of the TNF family (BAFF), and a proliferation-inducing ligand (APRIL), two members of the tumour necrosis factor (TNF) superfamily, beyond playing a significant role in normal B-cell development, promote survival and proliferation of malignant B cells. Both ligands interact with 3 receptors: BAFF-R, specific to BAFF, and TACI and BCMA which are shared by both BAFF and APRIL. Here we wished to investigate the potential role of these proteins in resistance of acute myeloid leukaemia (AML) blasts to apoptosis. We found that the levels of both mRNA and proteins of APRIL, BAFF and their receptors were expressed in leukaemic cells of 24 newly diagnosed, untreated AML patients. We also demonstrated that patients who did not further respond to induction therapy (NR) presented with significantly higher baseline APRIL and BAFF expression on AML blasts as compared to these subjects who, after induction, achieved complete remission (CR) following induction therapy. Moreover, we observed striking differences in baseline levels of BCMA between CR and NR patients as we did not find detectable expression of this receptor in the latter group of patients. Interestingly, we found that AML blasts collected at baseline from NR patients cultured in presence of exogenous BAFF and APRIL were significantly more resistant to spontaneous or drug-induced apoptosis as compared with cells derived from CR patients. Altogether, our data confirm that BAFF and APRIL signaling play important role in AML pathogenesis and susceptibility to cytotoxic therapy while measuring of BCMA expression on AML cells can become a novel prognostic factor for chemotherapy response.

Peripheral blood monocyte (PBM) subsets play different roles in inflammatory response and tissue remodelling. The aim of this study was to investigate how allergen challenge affects the number of circulating PBMs in Dermatophagoides pteronyssinus (Dp) allergic patients (Dp-APs). Among 34 Dp-APs challenged, in 22 patients significant bronchoconstriction was demonstrated [responders (Rs)], while in 12, only upper respiratory symptoms were seen [non-responders (NRs)]. Twelve healthy, non-atopic subjects were used as controls (HCs). Expression of CD14, CD16 and CCR4 was evaluated by flow cytometry on the whole-blood samples before (T(0) ), 6 h (T(6) ) and 24 h (T(24) ) after the challenge. Plasma concentrations of CCL2, CX3CL1 and CCL17 were evaluated using ELISA. At T(0) , the mean percentage of CD14++ CD16+ PBMs in Rs (35.4%; 95%CI 26.9-43.9%) was significantly greater than in HCs (14.6%; 95%CI 7.3-21.8%; P = 0.006) and in NRs (17.5%; 95%CI 9.6-25.4%; P = 0.001). The baseline number of CD14++ CD16+ PBMs correlated with airway hyper responsiveness (AHR) (r = -0.507; 95%CI -0.834 to -0.432, P < 0.001). At T(24) , the number of CD14++ CD16+ PBMs significantly decreased in Rs but not in NRs and the numbers inversely correlated with plasma CCL17 concentration. Changes in the number of circulating CD14++ CD16+ cells after Dp challenge correlated with AHR (r = 0.706, 95%CI 0.43-0.861; P < 0.001). In all subjects, the greatest expression of CCR4 was found on CD14++ CD16+ PBMs. Expansion of CD14++ CD16+ monocytes in the peripheral blood with subsequent mobilization of those cells after allergen challenge may facilitate the development of AHR in Dp-APs.

Urokinase plasminogen activator (uPA) and its inhibitor (PAI-1) are involved in tiisue remodeling and repair processes associated with acute and chronic inflammation. The aim of the study was to evaluate the effect of allergen challenge on concentration of uPA and PAI-1 in induced sputum of house dust mite allergic asthmatics (HDM-AAs). Thirty HDM-AAs and ten healthy persons (HCs)were recruited for the study. In 24 HDM-AAs bronchial challenge with Dermatophagoides pteronyssinus (Dp) and in 6 HDM-AAs sham challenege with saline were performed. In HDM-AAs sputum was induced 24 hours before (T0) and 24 hours (T24) after the challenge. Concentration of uPA and PAI-1 in induced sputum were determined using immunoenzymatic assays. At T0 in HDM-AAs mean sputum uPA (151 Âą 96 pg/ml) and PAI-1 (4341 Âą 1262 pg/ml) concentrations were higher than in HC (18.8 Âą 6.7 pg/ml; p=0.0002 and 596 Âą 180 pg/ml; p

Summary Glucocorticoids (GCS) are capable of stimulating the secretion of interleukin (IL)-10 by leucocytes; however, the potential of GCS to modulate leucocyte susceptibility to IL-10-mediated actions has not yet been studied. In the current paper, we performed a detailed cross-sectional analysis of IL-10 receptor (IL-10R) expression by CD4+ and CD8+ T cells, natural killer (NK) cells, monocytes and neutrophils. Next, we analysed the effects of short-term oral GCS treatment on surface IL-10R expression by various leucocyte subpopulations in asthmatic patients. All leucocyte subsets studied presented with substantial levels of surface IL-10R. The highest levels of IL-10R were found on monocytes, predominantly with CD142+CD16+ and CD14+CD16+ phenotypes, and on CD4+CD25high T cells. In contrast, levels of IL-10R on CD8+ T cells, NK cells and neutrophils were significantly lower and similar to each other in intensity. GCS treatment resulted in a significant decrease of IL-10R expression on all analysed peripheral blood leucocyte subsets. Our data suggest that down-regulation of IL-10R could counterbalance the otherwise suppressive action of GCS.

Background Recent studies in rodents revealed that regulatory T cells (T reg cells) with CD4+CD25+ phenotype can exert suppressive effects on experimentally-induced allergic airway inflammation and airway hyper-reactivity. It is unclear however, whether modulations of bronchoconstriction responses in human subjects might be related to T reg cells. We report here for the first time the changes in frequency of circulating lymphocytes with putative T reg cell phenotype (CD4+CD25+CD127low) in relation to bronchoconstriction phenotype following an intrabronchial allergen challenge. Material and methods Thirty-one house dust mite sensitive patients were challenged with Dermatophagoides pteronyssinus extract (Dp). Eleven isolated early responders (IER) were compared with nine dual (early and late) responders (DR) and to 11 non-responders (NR). Frequencies of peripheral blood CD4+CD25+CD127low lymphocytes were assessed in all groups of patients by using three-parameter flow cytometry before, and six and 24 h, after allergen inhalation. Results At baseline, frequencies of CD4+CD25+CD127low lymphocytes were not statistically different among NR, IER and DR. When all individuals were analyzed together, a statistically significant decrease in frequency of CD4+CD25+CD127low lymphocytes was observed 6 h after the bronchial challenge. Interestingly, such a pattern was found consistently only in NR, while IER and DR displayed varying responses resulting in a trend similar to that of NR. Twenty-four hours after the bronchial challenge, frequencies of CD4+CD25+CD127low lymphocytes in all groups tended to return to baseline values. Conclusions Our data indicate that bronchial allergen inhalation in sensitive patients (predominantly in those who did not develop significant bronchoconstriction) is associated with a decrease of proportion of peripheral lymphocytes with regulatory T cell phenotype.

Objective We evaluated blood concentrations of kynurenine pathway metabolites, natural and induced regulatory T cells (nTreg, iTreg), and Th17 cells in order to examine the activity of the kynurenine pathway and its relation to immune status in patients with pulmonary arterial hypertension (PAH). Methods Plasma concentrations of tryptophan, kynurenine, kynurenic acid, anthranilic acid, and 3-hydroxykynurenine were quantified in 26 patients with PAH (vs 30 healthy controls) at baseline and after 6 months, and assessed them in relation to clinical parameters, frequencies of lymphocyte subsets, and outcome. Results The PAH group presented higher concentrations of tryptophan (52.9 (IQR 46.3–57.5) vs 40.3 (35.2–46.3) µmol/L, p=0.00003), kynurenine 2.8 (2.4−3.4) vs 1.9 (1.5–2.3) µmol/L, p=0.000007), kynurenine/tryptophan ratio (0.051 (0.044–0.064) vs 0.043 (0.039–0.055), p=0.03), iTreg frequencies (10.5 (8.8–13.9)% vs 6.8 (5.2–9.5)%, p=0.002) and iTreg/Th17 (1.73 (1.2–2.8) vs 0.93 (0.61–1.27), p=0.003) together with lower ratios of kynurenic acid/kynurenine, 3-hydroxykynurenine/kynurenine, and anthranilic acid/kynurenine. Kynurenine concentrations and kynurenine/tryptophan ratio correlated positively with iTreg/Th17, and inversely with Th17 subsets, whereas kynurenic acid/kynurenine and anthranilic acid/kynurenine ratios correlated positively with Th17. Adverse outcomes occurred in 9 of 26 patients and they showed higher baseline concentrations of kynurenine (3.6 (2.8–4.3) vs 2.7 (2.1–3.2) µmol/L, p=0.033). Median kynurenine values ≥3.4 µmol/L (67% sensitivity, 94% specificity) identified patients with a worse clinical course. Conclusions PAH is characterised by upregulated tryptophan metabolism and enhanced biosynthesis of kynurenine. Elevated kynurenine concentration is associated with an adverse clinical course. Dysregulated immunity, delineated by Treg-Th17 imbalance, is directly related to diverse activation of the kynurenine pathway, indicating the potential interplay between kynurenines and the immune system in PAH.

Recently, murine hematopoietic progenitor stem cells (HSCs) and very small embryonic-like stem cells (VSELs) were demonstrated to express receptors for sex hormones including follicle-stimulating hormone (FSH). This raised the question of whether FSH therapy at clinically applied doses can mobilize stem/progenitor cells in humans. Here we assessed frequencies of VSELs (referred to as Lin−CD235a−CD45−CD133+cells), HSPCs (referred to as Lin−CD235a−CD45+CD133+cells), and endothelial progenitor cells (EPCs, identified as CD34+CD144+, CD34+CD133+, and CD34+CD309+CD133+cells) in fifteen female patients subjected to the FSH therapy. We demonstrated that FSH therapy resulted in statistically significant enhancement in peripheral blood (PB) number of both VSELs and HSPCs. In contrast, the pattern of responses of EPCs delineated by different cell phenotypes was not uniform and we did not observe any significant changes in EPC numbers following hormone therapy. Our data indicate that FSH therapy mobilizes VSELs and HSPCs into peripheral blood that on one hand supports their developmental origin from germ lineage, and on the other hand FSH can become a promising candidate tool for mobilizing HSCs and stem cells with VSEL phenotype in clinical settings.

Muscular dystrophies (MD) are heterogeneous group of diseases characterized by progressive muscle dysfunction. There is a large body of evidence indicating that angiogenesis is impaired in muscles of MD patients. Therefore, induction of dystrophic muscle revascularization should become a novel approach aimed at diminishing the extent of myocyte damage. Recently, we and others demonstrated that administration of granulocyte colony-stimulating factor (G-CSF) resulted in clinical improvement of patients with neuromuscular disorders. To date, however, the exact mechanisms underlying these beneficial effects of G-CSF have not been fully understood. Here we used flow cytometry to quantitate numbers of CD34+ cells, endothelial progenitor cells, and different monocyte subsets in peripheral blood of pediatric MD patients treated with repetitive courses of G-CSF administration. We showed that repetitive cycles of G-CSF administration induced efficient mobilization of above-mentioned cells including cells with proangiogenic potential. These findings contribute to better understanding the beneficial clinical effects of G-CSF in pediatric MD patients.

MicroRNAs (miRNAs) are currently experiencing a renewed peak of attention not only as diagnostics but also especially as highly promising novel targets or tools for clinical therapy in several different malignant diseases. Moreover, the recent discovery of competing endogenous RNAs (ceRNAs) as novel miRNA-regulators has contributed exciting insights in this regard. Therefore, this review summarizes and discusses the latest findings on (1) how miRNAs have become therapeutic targets of diverse synthetic antagonists, (2) how novel endogenous regulators of miRNAs such as ceRNAs or pseudogenes could emerge as therapeutics scavenging oncogenic miRNAs and (3) how miRNAs themselves are already, and will increasingly be, used as therapeutics. Recent advances on the importance of miRNA-target affinity and the subcellular localization of miRNAs are also discussed. The potential of these developments in different tumor entities and particular hallmarks of cancer such as metastasis, disease progression, interactions with the tumor microenvironment, or cancer stem cells are equally highlighted.

INTRODUCTION Recent studies in a mouse model of chronic lymphocytic leukemia (CLL) demonstrated that inhibition of the programmed death receptor 1 (PD‑1)–PD‑L1 axis resulted in correction of leuke‑ mia‑induced CD8 + T cell‑related immune dysfunction and protected mice against CLL development. However, it remains unclear whether CLL development and progression can be also associated with CD4 + T cells expressing PD‑1. OBJECTIVES We aimed to analyze whether a quantitative assessment of CD4 + PD‑1 + T cells performed at the time of diagnosis can have prognostic significance in patients with CLL. PATIENTS AND METHODS We examined 56 patients with newly diagnosed CLL at different stages of the disease. The quantitative assessment of PD‑1‑expressing CD4 + T cells was performed in all patients, using multicolor flow cytometry. RESULTS We demonstrated that CLL patients with an advanced (high and intermediate risk) stage had a significantly higher number of CD4 + PD‑1 + T cells compared with subjects with low‑grade disease. Importantly, we showed that the number of PD‑1‑expressing CD4 + T cells in the peripheral blood of patients referred for immediate treatment due to the advanced stage of the disease was significantly higher compared with subjects on watchful waiting. Finally, we found that treatment‑naive patients with higher numbers of CD4 + PD‑1 + T cells at baseline showed a significantly shortened time to the first treatment compared with patients with a low number of CD4 + PD‑1 + T cells. CONCLUSIONS Our study showed that the quantative assessment of CD4 + PD‑1 + T cells in peripheral blood using flow cytometry can facilitate prognostication of patients with newly diagnosed CLL.

MicroRNAs now not only represent a significant mechanism for post-transcriptional gene regulation, but have come to be appreciated as molecules with far reaching tentacles affecting diverse processes and pathologies by modulating amongst others, cellular gene expression, epigentic mechanisms, complex signaling cascades, cell-cell communication, the immune system and microenvironmental interactions between several cell types, tissues and organ systems. In this review, we systematically reflect on the impact of miRNAs on all types of benign and malignant human stem cells, looking at the roles they play in maintaining or changing the stem cell state, and review how aberrations of their expression and function within diverse types of stem cells orchestrate carcinogenesis and metastasis. As a conclusion, we consider it striking to see how similar some miR-driven mechanisms are between different types of stem cells and cancer cells, and how this might support hypotheses of miR-driven embryologic pathway reactivation in metastasis or propose putative functions of miRs in important novel cross-topic fields such as obesity and cancer.

Background Recently, identification of CD25 (interleukin-2 receptor alpha) expression on leukemic blasts was correlated to early treatment failure and unfavorable outcome in acute myeloid leukemia (AML) patients. Here we wished to determine whether quantification of CD25 on peripheral blood CD4+ T cells could improve prognostication in newly diagnosed AML patients. Methods The mean fluorescence intensity (MFI) of CD25 expression and frequencies of peripheral blood CD4+ T cells with varying levels of CD25 and CD127 expression were assessed by flow cytometry in all studied individuals. Results Using univariate (unadjusted) and multivariate (adjusted) analyses we demonstrated that detection of high pretreatment CD25 expression on circulating CD4+ T cells was associated with significantly decreased survival rate of AML patients subjected to standard induction chemotherapy. These associations held true for both entire group of analyzed AML patients and different subgroups of patients identified by presence or absence of favorable and adverse molecular prognostic factors. Conclusions Our data indicate that quantification of CD25 expression on peripheral blood CD4+ T cells could become a novel, easily accessible method of shortened survival prognostication of AML patients subjected to standard cytotoxic therapy.

Targeted therapy of non-small cell lung cancer (NSCLC) demands a more accurate tumor classification that is crucial for patient selection in personalized treatment. MicroRNAs constitute a promising class of biomarkers and a helpful tool for the distinction between lung adenocarcinoma (AC) and squamous cell lung carcinoma (SCC). The aim of this study was to evaluate the impact of two different normalization strategies, using U6 snRNA and hsa-miR-103 as reference genes, on hsa-miR-205 and hsa-miR-21 expression levels, in terms of the classification of subtypes of NSCLC. By means of a quantitative real-time polymerase chain reaction (qRT-PCR) microRNA expression levels were evaluated in a classification set of 98 surgically resected NSCLC fresh-frozen samples, and validated findings in an independent set of 42 NSCLC samples. The microRNA expression levels were exploited to develop a diagnostic test using two data normalization strategies. The performance of microRNA profiling in different normalization methods was compared. We revealed the microRNA-based qRT-PCR tests to be appropriate measures for distinguishing between AC and SCC (the concordance of histologic diagnoses and molecular methods greater than 88%). Performance evaluation of microRNA tests, based on the two normalization strategies, showed that the procedure using hsa-miR-103 as reference target has a slight advantage (sensitivity 83.33 and 100% in classification and validation set, respectively) compared to U6 snRNA. Molecular tests based on microRNA expression allow a reliable classification of subtypes for NSCLC and can constitute a useful diagnostic strategy in patient selection for targeted therapy.

We investigated whether the choice of anticoagulant or the application of density gradient mononuclear cell isolation may account for conflicting published data regarding the levels of the scavenger receptors' expression in healthy individuals. We demonstrate that the detection of CD163, but not CD36, differs dramatically among the methods.

Due to the alarming spread of human immunodeficiency virus type 1 (HIV-1) infection worldwide, the development of a prophylactic vaccine is critical. Vaccine strategies for AIDS have included the use of structural and nonstructural HIV subunit proteins or peptides, naked DNA, bacterial and viral live vectors, or combinations of the above. Most of the vaccines developed thus far induce T-cell responses but are unable to induce neutralizing antibodies to the Env of primary HIV-1 isolates. The fact that a decrease in plasma viremia (9, 41) is concomitant with the appearance of virus-specific cytotoxic T lymphocytes (CTLs) and depletion of CD8+ T cells in simian immunodeficiency virus (SIV)-infected macaques or HIV-infected chimpanzees suggests that CD8+ T cells are able to partially control HIV-1/SIV replication (15, 36, 43, 52). Indeed, several “T-cell vaccines” based on DNA and live vectors confer protection from high-level replication of challenge viruses in rhesus macaques (2, 4, 8, 30, 31, 48). The extent of the decrease in viral replication induced by these vaccines is variable in nonhuman primates, and protection from disease appears to be dependent on the virulence of the virus used in the challenge experiments (28). The relative efficacy of poxvirus-based vaccine candidates with various degrees of attenuation has been extensively studied in rhesus macaques after challenge with SIV, SHIV, and HIV-2 isolates (1, 3, 8, 24, 25, 29-31, 48). These vaccine modalities elicit variable levels of cell-mediated immune response and prevent infection after challenge exposure to viruses with low virulence, such as HIV-2 (3, 25), and to other somewhat-attenuated SIV isolates (34, 35). Importantly, these vaccine modalities were also able to significantly reduce virus load after challenge with highly pathogenic SIV isolates (8, 29, 32, 48). An ALVAC-SIV vaccine encoding the gag, pol, and env genes of the SIVmac251 isolate was able to reduce plasma virus load during primary infection and conferred protection from CD4+ loss during both acute and chronic phases of infection after rectal exposure to a highly pathogenic SIVmac251 isolate (50). Among the pox vector-based vaccines, several ALVAC-based HIV-1 vaccines have been tested in phase I and II clinical trials and have been shown to be safe and immunogenic in humans (10, 14, 18, 21, 27). Whether the immunogenicity of these vaccine candidates will be sufficient to protect humans from HIV-1 remains unknown (5, 11, 44). The ongoing human phase III trial in Thailand will provide key information in this regard. Here, we designed a study to assess whether systemic immunization with recombinant canarypox expressing either HIV-1 gp120 or gp160 followed by a boost with either purified gp120 or gp140 proteins would confer protection after mucosal challenge with the pathogenic SHIVKU2. We also investigated whether the addition of the Tat protein of HIV-1 as an immunogen could provide better protection, since a few studies have demonstrated that immunization of macaques with Tat protein either alone or as a Nef-Tat fusion protein in a multicomponent subunit vaccine confers an advantage in a SHIV89.6P model (12, 13, 56) but not in other studies (53). Here, we found that immunization of macaques with this type of ALVAC-based vaccine formulation elicited both antibody and cellular responses and significantly decreased plasma viremia and CD4+ T-cell loss after rectal exposure to the SHIVKU2 isolate. Tat protein immunization had no additive effect on the reduction of virus load in vaccinated rhesus macaques, as also observed by others (42, 46, 53).

IL-2, the first cytokine discovered with T cell growth factor activity, is now known to have pleiotropic effects on T cells. For example, it can promote growth, survival, and differentiation of Ag-selected cells, or facilitate Ag-induced cell death of T cells when Ag persists, and in vivo, it is thought to contribute to the regulation of the size of adaptive T cell response. IL-2 is deficient in HIV-1 infection and has been used in the management of HIV-1-infected individuals undergoing antiretroviral therapy. In this study, we investigated how continuous low-dose IL-2 affected the CD4+ and CD8+ T cell response induced by two inoculations of a canarypox recombinant SIV-based vaccine candidate in healthy macaques chronically infected with SIVmac251. These macaques had normal levels of CD4+ T cells at the beginning of antiretroviral therapy treatment. Vaccination in the presence of IL-2 significantly augmented Gag-specific CD8+ T cell responses, but actually reduced Gag-specific CD4+ T cell responses. Although IL-2 at low doses did not change the overall concentration of circulating CD4+ or CD8+ T cells, it expanded the frequency of CD4+CD25+ T cells. Depletion of the CD4+CD25+ T cells in vitro, however, did not result in a reconstitution of Gag-specific CD4+ responses or augmentation of SIV-specific CD8+ T cell responses. Thus, we conclude that the decrease in virus-specific CD4+ T cell response may be due to IL-2-promoted redistribution of cells from the circulation, or due to Ag-induced cell death, rather than suppression by a T regulatory population.

Infection with human immunodeficiency virus or simian immunodeficiency virus (SIV) induces virus-specific CD8+T cells that traffic to lymphoid and nonlymphoid tissues. In this study, we used Gag-specific tetramer staining to investigate the frequency of CD8+T cells in peripheral blood and the central nervous system of Mamu-A*01-positive SIV-infected rhesus macaques. Most of these infected macaques were vaccinated prior to SIVmac251 exposure. The frequency of Gag181-189CM9 tetramer-positive cells was consistently higher in the cerebrospinal fluid and the brain than in the blood of all animals studied and did not correlate with either plasma viremia or CD4+-T-cell level. Little or no infection in the brain was documented for most animals by nucleic acid sequence-based amplification or in situ hybridization. These data suggest that this Gag-specific response may contribute to the containment of viral replication in this locale.