Milena Dabrowska

Enhanced frequencies of CD14++CD16+, but not CD14+CD16+, peripheral blood monocytes in severe asthmatic patients

CD16+ monocytes are expanded in various inflammatory conditions. Recently it was reported that CD16+ monocytes can be divided into two subsets with contrasting potential of modulating inflammatory responses, namely CD14++CD16+ and CD14+CD16+ monocytes. Here, we characterized and quantified CD14++CD16+ and CD14+CD16+ monocyte subsets in asthmatic patients in the context of severity of disease and different treatment options. Subjects included seventeen severe asthmatics and eighteen moderate asthmatics treated with moderate-to-high doses of inhaled glucocorticosteroids (GCS), twenty nine steroid-naive mild asthmatics and fifteen healthy controls. First, we demonstrated that CD14++CD16+ monocytes, in contrast to CD14+CD16+ monocytes, present significantly higher expression of anti-inflammatory molecule CD163. The frequency of CD14++CD16+, but not CD14+CD16+ monocytes, was significantly higher in patients with severe asthma as compared to mild and moderate asthmatics. However, the frequency of both CD16+ monocyte subsets did not correlate directly with exhaled nitric oxide levels. Short-term administration of oral GCS in patients with exacerbations resulted in a preferential decrease of CD14+CD16+ monocytes. Our study indicates that CD14++CD16+ and CD14+CD16+ monocyte subsets in asthmatics are differentially modulated by both the inflammatory process and GCS treatment.

Reactive oxygen species activate mitogen-activated protein kinases in pancreatic acinar cells.

It has been recently reported that kinases that belong to the mitogen-activated protein kinase (MAPK) family are rapidly activated by cholecystokinin (CCK) in rat pancreas both in vitro and in vivo. It is known that reactive oxygen species (ROS) play an important role in the pathogenesis of acute pancreatitis induced by supraphysiologic stimulation with CCK analogue, cerulein. The aim of our study was to evaluate whether MAPKs are activated by ROS in pancreatic acini. The activity of MAPK, c-Jun amino-terminal kinase (JNK), and p38 MAPK was determined in isolated rat pancreatic acinar cells by means of Western blotting, with the use of specific antibody that recognizes active, dually phosphorylated kinases. Incubation of acini with ROS donors, hydrogen peroxide (H2O2) and/or menadione (MND), strongly activated all three kinases. Activation of these kinases by ROS, but not by CCK, was substantially inhibited by pretreatment of acini with antioxidant N-acetylo-l-cysteine (NAC). Whereas CCK-induced activation of MAPK or JNK was totally or partially blocked by protein kinase C (PKC) inhibitor GF-109203X, ROS-induced activation of MAPK, JNK, and p38 MAPK was PKC independent. In conclusion, ROS strongly activate MAPK, JNK, and p38 MAPK in pancreatic acinar cells. It may be of importance in acute pancreatitis, because ROS are involved in the pathogenesis of this disease.

Frequencies of circulating CD4+CD25+CD127low cells in atopics are altered by bronchial allergen challenge.

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.

Expression of interleukin 4 receptors in bronchial asthma patients who underwent specific immunotherapy.

BACKGROUND Interleukin (IL) 4 and IL-13 are crucial cytokines for the development of allergic reactions and have been shown to modulate the function of monocytes and macrophages. OBJECTIVES To evaluate the expression of IL-4Rs on peripheral blood monocytes and in the serum of patients with bronchial asthma who underwent specific immunotherapy (SIT). METHODS The study was performed on 17 asthma patients with a typical clinical history and positive skin prick test results to Dermatophagoides pteronyssinus allergens. Five asthma patients who declined SIT were used as a comparator control group. Ten healthy persons served as negative controls. Flow cytometry analysis was performed on the whole blood samples using labeled monoclonal antibodies against CD14 and CD36 monocyte markers and against the CD124 alpha chain of IL-4R. The serum levels of soluble IL-4R were evaluated using an immunoenzymatic assay. RESULTS Compared with controls, bronchial asthma patients before SIT had a higher mean +/- SD percentage of CD14-positive cells that coexpressed CD124 (3.5% +/- 1.8% vs 18.6% +/- 7.9%; P < .01). After SIT, the mean +/- SD percentage of CD14 cells coexpressing CD124 decreased to 8.1% +/- 5.1%, which was significantly lower than before SIT (P < .01) but still significantly higher than in controls (P = .01). Changes in CD124 expression were associated with up-regulation of CD14 and down-regulation of CD36 expression on peripheral blood monocytes, suggesting that IL-4/IL-13-mediated signaling may be important for regulation of monocyte phenotype and function in asthma patients receiving SIT. CONCLUSIONS Even short courses of SIT are associated with a decrease in IL-4R expression on peripheral blood monocytes, which may cause decreased IL-4/IL-13-mediated effects in patients who undergo SIT.

Allergen Challenge Differentially Affects the Number of Circulating Monocyte Subsets

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 (T0), 6 h (T6) and 24 h (T24) after the challenge. Plasma concentrations of CCL2, CX3CL1 and CCL17 were evaluated using ELISA. At T0, 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 T24, 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.

Oral glucocorticoid treatment decreases interleukin-10 receptor expression on peripheral blood leucocyte subsets

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.

Monocyte Subsets and Natural Killer Cells in Acute Pancreatitis

Background: Alteration of the immune system is one of the major mechanisms responsible for complications in severe acute pancreatitis (AP). The aim of our study was to provide a complex evaluation of peripheral blood monocyte subsets, natural killer cells (NK cells) and cytotoxic T lymphocytes in patients with different severity forms of AP. Methods: 20 patients with mild AP and 15 with severe AP (S-AP) were included in our study. Peripheral blood mononuclear cells were studied on days 1–3, 5, 10 and 30, by means of flow cytometry. Results: In peripheral blood of patients with pancreatitis, we found a marked increase in total monocyte count. In S-AP, circulating monocytes were significantly activated, which was presumed from increased expression of HLA-DR, CD54, CD69 and CD25. Concurrent increased expression of CD95 (FasR) may indicate enhanced susceptibility of these cells to apoptosis. In patients with S-AP, a dramatic depletion of circulating NK cells (CD16/56 and CD3– CD8+) was found along with a reduction of circulating CD3+ CD8+ lymphocytes (cytotoxic T lymphocytes). Conclusion: Our findings suggest profound disturbances of innate cellular immunity in patients with S-AP.

Effective Mobilization of Very Small Embryonic-Like Stem Cells and Hematopoietic Stem/Progenitor Cells but Not Endothelial Progenitor Cells by Follicle-Stimulating Hormone Therapy

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.

Does prematurity affect platelet indices

PURPOSE The current study objective was to compare blood platelet indices in preterm newborns (PTN) and full term newborns (FTN). MATERIALS AND METHODS We introduced to our study 51 PTN (25 females, 26 males) and 55 FTN (25 females, 30 males). Platelet indices were estimated in blood samples collected from the umbilical artery. RESULTS PTN demonstrated a decreased count of blood platelets (197 x 103/microL) as compared to FTN (287 x 103/microL), p=0.0001. Platelet hematocrit (PCT) also showed substantial differences in both groups (PTN=0.16% vs. FTN=0.22%; p=0.001). Mean platelet volume (MPV) was found to be nearly the same (PTN=8.02 fl, FTN=7.79 fl). Platelet distribution width (PDW) was higher in PTN (50.64%) than in FTN (46.54%), p=0.021. Large platelet count (LPLT) was diminished in PTN (5.23%) in comparison with FTN (6.12 %). CONCLUSIONS A decreased count of blood platelets, platelet hematocrit and increased platelet distribution width may result from a low gestational age or a dysfunction of megakaryocytes and the placenta. Blood platelet indices may be vital in the diagnosis of haemostatic disorders.

Activity of the kynurenine pathway and its interplay with immunity in patients with pulmonary arterial hypertension

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.