Effect of Dermatophagoides pteronyssinus extract on the expression of genes involved in inflammation and tissue remodeling by peripheral blood mononuclear cells of allergic asthma patients.

PURPOSE: House dust mite allergy constitutes a risk factor for asthma development and is associated with a faster decline of lung function in allergic asthmatics (AAs). To evaluate the effect of Dermatophagoides pteronyssinus (Dp) allergens on the expression of genes involved in inflammation and tissue remodeling by peripheral blood mononuclear cells (PBMCs) isolated from the blood of AAs. MATERIALS AND METHODS: The cells from AAs, allergic rhinitis without asthma patients (ARs), and healthy controls (HCs) were cultured in the presence of Dp, lipopolysaccharide (LPS), or without any stimulation. The expression of 84 genes was evaluated using a low-density microarray whereas, the quantitative expression analysis of selected genes was performed using a real-time polymerase chain reaction. The concentration of selected proteins in the cell culture supernatants was assessed using ELISA. RESULTS: Stimulation of PBMCs with Dp and LPS resulted in a significant upregulation of 8 and 15 among 84 studied genes, respectively. The greatest upregulation was observed for CCL2 and CCL3 using Dp and LPS, respectively. In comparison with HCs, in AAs, significantly increased upregulation of CCL2 in response to Dp was found. The secretion of CCL2 and CCL3 by PBMCs reflected the pattern of gene expression at the mRNA level. The mean Dp-stimulated secretion of CCL2 by PBMCs of ARs was less than in AAs (p â€‹< â€‹0.01), both being notably greater than in the HCs (p â€‹< â€‹0.01). CONCLUSION: Rapid and potent upregulation of CCL2 expression by PBMCs in response to Dp may constitute an important contribution to the development of allergic asthma.

Systemic corticosteroid therapy augments ex vivo release of sCD163 by peripheral blood monocytes of asthmatic patients.

INTRODUCTION: The CD163 is exclusively expressed by mononuclear phagocytes as a transmembrane protein, which synthesis is regulated by anti- and pro-inflammatory signals. After shedding from the cell surface it exists in body fluids as a soluble protein (sCD163) which exerts anti-inflammatory effects. AIM: To evaluate serum concentration and ex vivo production of sCD163 by peripheral blood mononuclear cells (PBMC) in asthmatic patients treated with inhaled (ICS) or oral corticosteroids (OCS). MATERIAL AND METHODS: The study was performed on 35 allergic asthma patients (AAs) including 15 treated with ICS (ICS-AAs), 10 with OCS (OCS-AAs), 10 during asthma exacerbation (EX-AAs) before OCS had been started and 13 non-atopic healthy subjects (HCs) as a control group. PBMC were cultured in vitro for up to 144 h. The concentration of sCD163 in serum and the culture supernatants was evaluated with ELISA. RESULTS: The greatest serum sCD163 concentration was demonstrated in EX-AAs, which was significantly greater than that in other studied subgroups. The concentration of sCD163 in PBMC culture supernatants was greater in AAs than in HCs (p = 0.006). Among individual asthma subgroups the greatest concentration of sCD163 was demonstrated in PBMC culture supernatants of OCS-AAs, which was significantly greater than in ICS-AAs (p < 0.001) and EX-AAs (p < 0.001), both being significantly greater than in HCs (p < 0.001). CONCLUSIONS: In AAs, enhanced capability of PBMCs to release sCD163 may be at least partially responsible for the anti-inflammatory effects of systemic corticosteroid therapy.

Synergistic effect of Dermatophagoides pteronyssinus allergens and dexamethasone on the expression of CD163 by peripheral blood mononuclear cells.

BACKGROUND: The CD163 is a marker of monocyte/macrophage anti-inflammatory function. Its soluble form (sCD163) also exert anti-inflammatory activities including inhibition of T cell proliferation. OBJECTIVE: To evaluate the effect of dexamethasone (Dx) and Dermatophagoides pteronyssinus (Dp) on ex vivo production of sCD163 by peripheral blood mononuclear cells (PBMCs). METHODS: PBMCs from 26 allergic asthma patients (AAs) and 12 non-atopic healthy controls (HCs) were cultured with Dp, Dx, Dp + Dx or without any stimulation for up to 144 h (T144). Concentration of sCD163, interleukin (IL) -6 and IL-10 in PMBC culture supernatants was evaluated using ELISA. The mRNA expression of CD163 by PBMCs was estimated using quantitative PCR (qPCR). RESULTS: At T144 the median concentration of CD163 in unstimulated PBMC cultures of AAs was greater than that in HCs (p = 0.008). Concomitant application of Dp and Dx resulted in a synergistic effect reflected by a dramatic increase of sCD163 concentration both in HCs (p = 0.0002) and AAs (p < 0.0001). Also a synergistic effect of Dp and Dx on CD163 mRNA expression was seen at T24 and T48 but not at T6 or T12. Among asthmatic patients the effect of Dx on sCD163 production was attenuated in severe in comparison to mild-to-moderate AAs (p = 0.0007). Moreover, Dp-induced production of IL-6 but not IL-10 was inhibited by Dx (p < 0.0001). Inhibition of IL-10 decreased sCD163 concentration by more than 50%. CONCLUSIONS: Dx-triggered upregulation of anti-inflammatory CD163 expression by monocytes is synergistic with endogenous mechanisms involved in the resolution of Dp-induced inflammation. This effect is impaired in severe asthma patients.

The efficacy and safety of pomalidomide in relapsed/refractory multiple myeloma in a "real-world" study: Polish Myeloma Group experience.

BACKGROUND: Patients with relapsed/refractory multiple myeloma (RRMM) have poor prognosis. Pomalidomide is an immunomodulatory compound that has demonstrated activity in MM patients with disease refractory to lenalidomide and bortezomib. OBJECTIVES: Participants of clinical trials are highly selected populations; therefore, the aim of this study was to present observations from real practice that might provide important information for practitioners. PATIENTS AND METHODS: We analyzed retrospectively 50 patients treated with pomalidomide in 12 Polish sites between 2014 and 2017. Median age was 63 years, median time since diagnosis 4.5 years and median number of prior regimens 4. RESULTS: The overall response rate was 39.1%. Median progression-free survival (PFS) and overall survival (OS) were 10.0 and 14.0 months, respectively. Previous treatment with immunomodulatory drugs, bortezomib or stem cell transplant had no impact on PFS and OS. Most frequent grade 3/4 treatment-emergent adverse events were hematologic (neutropenia 24.0%, thrombocytopenia 10.0%, anemia 8.0%). Most common grade 3/4 non-hematologic toxicities were respiratory tract infection (14.0%) and neuropathy (4.0%). CONCLUSIONS: This real-world data have confirmed that pomalidomide is an active drug in RRMM and support results of published clinical trials and other real-world studies.

Cardiovascular Risk Factors after Childhood Cancer Treatment Are Independent of the FTO Gene Polymorphism?

The study objective was to assess the prevalence of cardiovascular disease risk factors in patients treated for childhood cancer (N = 101) and to determine the involvement of clinical (cancer type and therapy) and/or genetic (FTO gene rs9939609 polymorphism) factors. Anthropometric features, laboratory findings, and standardized osteodensitometric indices (fat and lean mass) were considered. Overweight/obesity was found in 17.82% of the patients; however, central adiposity was found in as many as 42.5%. At least one abnormality in lipid metabolism was observed in 35.6%. Densitometry revealed elevated levels of fat mass in 44.55% of the patients. None of the parameters studied were associated with the FTO gene polymorphism. Standardized waist circumference was significantly higher in patients treated for leukemia than those treated for solid tumors (p = 0.04). Our findings indicate a high rate of central adiposity among childhood cancer survivors, especially leukemia patients. The prevalence of risk factors of cardiovascular disease after anticancer therapy is not FTO gene polymorphism-dependent.

Histone deacetylases affect transcriptional regulation of CCL2 and CXCL8 expression by pulmonary fibroblasts in vitro.

INTRODUCTION: Chemokines have been shown to play an important role in tissue remodeling and fibrosis in the respiratory system. In this study we wanted to evaluate the mechanisms, which regulate the expression of selected chemokines by pulmonary fibroblasts in vitro. MATERIAL AND METHODS: Pulmonary fibroblasts were cultured with and without bacterial lipopolysaccharide (LPS) for 6 hours. In addition some of the cultures were pre-treated with histone deacetylase inhibitor Trichostatin A (TSA). Real-time PCR reaction was performed to estimate the expression of chemokines CCL2, CCL3 and CXCL8. RESULTS: In unstimulated cultures detectable expression of CCL2 and CXCL8 was observed, while CCL3 expression could not be detected. After stimulation with LPS, TSA and both agents together CCL2 expression rose by 1.52, 1.62 and 1.8 times in comparison to control cultures respectively. CXCL8 mRNA expression levels after stimulation with LPS, TSA and LPSTSA increased by 1.53, 1.91 and 2.4 times accordingly. CONCLUSION: Epigenetic mechanisms related to histone acetylation affects transcriptional regulation of CCL2 and CXCL8 expression by pulmonary fibroblasts. Those mechanisms may play a role in tissue repair and pathologic remodeling.

The Effect of Afatinib Treatment in Non-small Cell Lung Cancer Cells.

BACKGROUND: Non-small cell lung cancer (NSCLC) is a very aggressive and histologically heterogeneous type of lung cancer. The main problem in the treatment of NSCLC is chemoresistance and metastasis. Compared to other malignant tumors, many molecular mechanisms are dysfunctional in NSCLC. Epidermal growth factor receptor (EGFR) is one of the most frequently mutated genes in NSCLC. MATERIALS AND METHODS: We investigated the effect of afatinib the against A549 NSCLC cell line, because it is active against mutated EGFR. Moreover, we aimed to investigate the mRNA level of HSPA5 (one of the heat shock proteins that can contribute in the down-regulation of the EGF-signaling pathway) before and after afatinib treatment. RESULTS: Afatinib treatment induced apoptosis and decreased levels of HSPA5 mRNA in cancer cells. CONCLUSION: Advanced analysis, might be helpful in understanding the mechanisms of relations between tyrosine kinase inhibition of EGFR and HSPA5 in lung cancer cells.

The arachidonate 5-lipoxygenase activating protein gene polymorphism is associated with the risk of scleroderma-related interstitial lung disease: a multicentre European Scleroderma Trials and Research group (EUSTAR) study.

Objectives: The arachidonate 5-lipoxygenase activating protein (ALOX5AP) regulates synthesis of leukotrienes (LTs), which are important mediators of inflammation and connective tissue remodelling. The aim of this study was to evaluate if single nucleotide polymorphisms (SNPs) of ALOX5AP confer risk of SSc and/or SSc-related organ involvement. Methods: Seven SNPs of ALOX5AP (rs17222814, rs17216473, rs10507391, rs4769874, rs9551963, rs9315050 and rs7222842) were genotyped in a cohort of 977 patients with SSc and 558 healthy controls from centres collaborating within the European Scleroderma Trials and Research group. In 22 SSc patients, concentrations of cysteinyl LTs and LT B4 (LTB4) were measured in the supernatants of ionophore-stimulated peripheral blood mononuclear cells (PBMCs) by means of commercially available enzyme immunoassay kits. Results: Significant association was found between rs10507391 polymorphism (T/A) of ALOX5AP and the risk of SSc [odds ratio (OR) 1.27 (95% CI 1.07, 1.50), P < 0.05 vs controls], the presence of SSc-related interstitial lung disease on high-resolution CT of the lungs [OR 1.45 (95% CI 1.17, 1.79), P < 0.05 vs patients without SSc-related interstitial lung disease] as well as with restrictive ventilatory defect [forced vital capacity <70% of predicted; OR 1.51 (95% CI 1.16, 1.97), P < 0.05 vs SSc patients without pulmonary restriction]. PBMCs from SSc carriers of rs10507391 allele A synthesized greater amounts of cysteinyl LTs as compared with SSc patients with rs10507391 TT genotype ( P < 0.05). Synthesis of LTB4 did not differ significantly between the two groups. Conclusion: The results of our study indicate that the genetic variants of ALOX5AP might play a role in the development of SSc-related pulmonary fibrosis.

Soluble CD163 modulates cytokine production by peripheral blood mononuclear cells stimulated by Dermatophagoides pteronyssinus allergens in vitro.

PURPOSE: The CD163 is a scavenger receptor expressed exclusively on monocytes/macrophages which has been shown to exert anti-inflammatory effects. The aim of this study was to evaluate the effect of exogenous sCD163 on production of selected cytokines by peripheral blood mononuclear cells (PBMC) of house dust mite allergic patients (AAPs) stimulated in vitro with Dermatophagoides pteronyssinus (Dp) allergens. PATIENTS AND METHODS: The study was performed in 24 AAPs and 12 healthy control subjects (HCs). Peripheral blood mononuclear cells were cultured for up to 144h (T144) in the presence of Dp extract with or without sCD163. Concentration of interleukin (IL) - 10, IL-13 and transforming growth factor beta (TGF-ß) was evaluated in the cell culture supernatants using ELISA. Expression of the selected cytokines was evaluated in cell culture lysates using Taqman-based real time polymerase chain reaction (RT-PCR). RESULTS: Dp-stimulated PBMC from AAPs released more IL-10 and IL-13 than those from HCs. The greatest up-regulation of IL-10 expression was seen at T6, while that of IL-13 was delayed. Soluble CD163 augmented production of IL-10 in response to Dp stimulation. No significant effect of sCD163 on production of IL-13 and IL-10 by PBMC of HCs could be demonstrated. CONCLUSIONS: In AAPs sCD163 modulates the immune response to Dp allergens potentiating anti-inflammatory, homeostatic mechanisms.

The role of 12/15-lipoxygenase in production of selected eicosanoids in allergic airway inflammation.

PURPOSE: To evaluate the role of 12/15-lipoxygenase (LOX) in regulation of synthesis of selected eicosanoids in mice sensitized and challenged with Dermatophagoides pteronyssinus (Dp) allergen extract. MATERIALS AND METHODS: Both C57Bl and 12/15-LOX knockout mice were sensitized by 2 intraperitoneal injections and subsequently challenged by inhalation with Dp allergen extract. Sham sensitized and challenged mice were used as controls. Samples of bronchoalveolar lavage (BAL) were used for assessment of prostaglandin E2 (PGE2), cysteinyl leukotreienes (cysLT), lipoxin A4 (LXA4) and 15-hydroxyeicosatetraenoic acid (15-HETE) concentration using ELISA method. Whole lung samples were used for isolation of RNA and evaluation of selected genes involved in eicosanoid metabolism, including cyclooxygenase-2 (COX-2), 12/15-LOX, 5-LOX and 5-LOX activated protein (FLAP). RESULTS: Allergen-induced airway inflammation was associated with significant (9-fold, 95% CI 8.068-9.932-fold; p<0.05) up-regulation of 12/15-LOX in wild type but not in the 12/15-LOX knockout mice in which 12/15-LOX mRNA remained undetectable. Lack of 12/15-LOX was associated with significant attenuation of production of 15-HETE in response to allergen challenge. On the contrary, the greatest up-regulation of COX-2 after allergen exposure was demonstrated in the 12/15-LOX knockout mice (4.3-fold vs sham group) and was significantly greater than in the wild type counterparts (5.185-fold, 95% CI 4.723-6.309-fold; p<0.05 vs wild type mice). Also, allergen challenged 12/15-LOX knockout mice were characterized by greater production of PGE2 and cysLT. CONCLUSION: The 12/15-LOX plays an important role in the metabolism of eicosanoids in response to allergen-induced airway inflammation.

Polymorphism of the FTO Gene Influences Body Weight in Children with Type 1 Diabetes without Severe Obesity.

The objective was to compare the impact of clinical and genetic factors on body mass index (BMI) in children with type 1 diabetes (T1DM) without severe obesity. A total of 1,119 children with T1DM (aged 4-18 years) were qualified to take part in the study. All children were genotyped for variants of FTO, MC4R, INSIG2, FASN, NPC1, PTER, SIRT1, MAF, IRT1, and CD36. Results. Variants of FTO showed significant association with BMI-SDS in the T1DM group. The main factors influencing BMI-SDS in children with T1DM included female gender (P = 0.0003), poor metabolic control (P = 0.0001), and carriage of the A allele of the FTO rs9939609 gene (P = 0.02). Conclusion. Our research indicates, when assessing, the risk of overweight and obesity carriage of the A allele in the rs9939609 site of the FTO gene adds to that of female gender and poor metabolic control. This trial is registered with ClinicalTrials.gov (NCT01279161).

[Polymorphism rs9939609 of FTO gene is related to the body mass index in children from Podlaskie voievodship]. / Polimorfizm rs9939609 genu FTO jest zwiazany ze wskaznikiem masy ciala dzieci z wojewodztwa podlaskiego.

INTRODUCTION: The presence of obesity and the features of metabolic syndrome plays a predictive role in cardiovascular diseases (CVD) in adults. It seems reasonable to seek new risk factors in the development of CVD. Defining the genetic background of obesity could help to select patients from a high risk group and help to introduce prevention and treatment, which, in consequence, lead to the lowering of morbidity and mortality. One of the genes probably related to the body weight is the Fat Mass and Obesity Associated Gene (FTO). THE AIM: of the study was an attempt to assess the relationship between the FTO polymorphism rs9939609 and body mass index in children from Podlaskie voievodship. MATERIAL AND METHODS: 405 children aged 4-18 were selected for the study. The examination included body mass index, waist circumference, blood pressure and lipid profile analysis. FTO rs9939609 polymorphism was assessed using a discrimination allele method with the application of ABI 7900HT Fast Real-Time PCR System. RESULTS: FTO rs9939609 polymorphism was related to the standarized body mass index and the AA genotype carriers had a higher risk of obesity. This polymorphism was also associated with waist circumference, systolic blood pressure and triglycerides concentration. It was not correlated with diastolic blood pressure and total HDL- and LDL-cholesterol concentrations. CONCLUSIONS: Our results demonstrate that rs9939609 FTO gene polymorphism is related to the body mass index in children. Our results should be confirmed in studies on a large cohort of healthy Polish children.

Increased production of a proliferation-inducing ligand (APRIL) by peripheral blood mononuclear cells is associated with antitopoisomerase I antibody and more severe disease in systemic sclerosis.

OBJECTIVE: A proliferation-inducing ligand (APRIL), a member of the tumor necrosis factor (TNF) family, plays a crucial role in the survival of peripheral B cells, and may contribute to the pathogenesis of systemic sclerosis (SSc) through upregulation of autoantibody production and maintenance of autoimmune phenomena. We evaluated the capacity of peripheral blood mononuclear cells from patients with SSc (SSc-PBMC) to produce APRIL; and investigated correlations between production of APRIL by SSc-PBMC and clinical and laboratory features of the disease. METHODS: PBMC from 20 patients with SSc and 14 healthy subjects were incubated in fetal calf serum-supplemented RPMI medium. APRIL levels were determined in cell culture supernatants by ELISA. RESULTS: PBMC from patients with SSc produced significantly more APRIL (961 ± 151 pg/ml/105 cells) than control PBMC (798 ± 219 pg/ml/105 cells; p < 0.01). In patients with SSc, increased production of APRIL was associated with the presence of diffuse skin involvement, scleroderma lung disease, peripheral vasculopathy, greater capillary damage on capillaroscopy, and presence of anti-topoisomerase I (anti-topo I) antibodies. Multivariate regression analysis revealed anti-topo I antibodies as the only independent predictor of high production of APRIL by PBMC. CONCLUSION: Production of APRIL is increased in SSc-PBMC and is associated with the presence of anti-topo I antibodies and more severe disease. Targeting the APRIL pathway might represent a therapeutic possibility for treatment of patients with SSc, in particular those with anti-topo I antibodies.

[Overweight, obesity and metabolic syndrome in children with type 1 diabetes melllitus]. / Nadwaga, otylosc i cechy zespolu metabolicznego u dzieci z cukrzyca typu 1.

INTRODUCTION: Obesity can be an additional risk factor for developing cardiovascular diseases in patients with diabetes. Aim of the study was the assessment of overweight, obesity and other elements of the metabolic syndrome in children with type 1 diabetes mellitus. MATERIAL AND METHODS: 300 children treated with insulin at least one year were enrolled in the study. In the examined group anthropometric data, data concerned with diabetes and additional laboratory tests including risk factors for cardiovascular diseases were assessed. RESULTS: The median age of the examined group was 13.7 years. The body mass deficiency was noted in 0.66%, normal body mass in 71.6%, overweight in 15.3% and obesity in 12.3%. The abdominal obesity was noted in 16.0% of children. The rise in the body weight between 3-6 months from the beginning of the insulin therapy and the present assessment was statistically significant. Children with normal weight had a better metabolic control in comparison to children with overweight/obesity. Girls had a higher rise in body mass index values between the time of diagnosis and the present investigation compared to boys. Higher values of blood pressure or hypertension were noted in 16.6% of children. Altogether in 25.3% of children some dyslipidemia was observed. The metabolic syndrome criteria were noted in: 28.0% - one criterion, 13.0% - two criteria, and 0.3% - three criteria. CONCLUSIONS: The population of children with type 1 diabetes is characterized by high frequency of overweight/obesity, abdominal obesity, dyslipidemia and hypertension. The features of metabolic syndrome are less frequent. It is worthwhile to monitor the risk for development of cardiovascular diseases in this group of children.