Dorota Trzybulska

Interleukin-17 and interleukin-23: importance in the pathogenesis of lung impairment in patients with systemic sclerosis

T cell abnormalities with a focus on Th17 cells have been associated with the pathogenesis of systemic sclerosis (SSc) and interstitial lung disease (ILD). The aim of this study was to evaluate serum levels of interleukin (IL)‐17, IL‐21 and IL‐23 in SSc patients and to assess their relationship with ILD‐SSc.

Serum Clara cell 16-kDa protein levels and lung impairment in systemic sclerosis patients

OBJECTIVE To assess clinical utility of serum Clara cell 16-kDa protein measurements in relation with staging system for systemic sclerosis associated interstitial lung disease. MATERIALS AND METHODS Serum levels of Clara cell 16-kDa protein were determined by ELISA in 28 systemic sclerosis patients and 30 healthy controls, and correlated with staging system for systemic sclerosis associated interstitial lung disease in systemic sclerosis patients. Lung involvement was assessed functionally (body plethysmography, diffusing capacity of the lung for carbon monoxide) and radiologically (an average disease extent on high resolution computed tomography of the lungs) in SSc patients. RESULTS We observed statistically significant differences in serum Clara cell 16-kDa protein levels between systemic sclerosis patients and healthy controls only in non-smokers. However, serum Clara cell 16-kDa protein concentrations were significantly elevated in patients with high resolution computed tomography extent >20% in comparison to patients with high resolution computed tomography extent <20% (p=0.01). They correlated positively with average disease extent on high resolution computed tomography (p=0.04), an extent of a reticular pattern on high resolution computed tomography (p<0.01), and negatively with a total lung capacity (p=0.03) and the results of the 6-min walk test (p<0.01). CONCLUSIONS Clara cell 16-kDa protein levels can be considered as a supplemental serum biomarker for systemic sclerosis associated interstitial lung disease.

Quantitative analysis of elastase and cathepsin G mRNA levels in peripheral blood CD14(+) cells from patients with rheumatoid arthritis.

In rheumatoid arthritis (RA) activity of serine proteases is an important factor contributing to destructive changes in the joints. The aim of this study was to compare elastase (ELANE) and cathepsin G (CTSG) mRNA levels in peripheral blood CD14(+) cells obtained from RA patients, healthy subjects (HS) and patients with osteoarthritis (OA). CD14(+) cells were isolated from peripheral blood by positive magnetic selection. The expression levels of ELANE and CTSG were determined by quantitative real-time PCR. ELANE mRNA expression was significantly higher in RA patients when compared to HS (p<0.001) and OA patients (p<0.001). The results suggest that in RA, peripheral blood CD14(+) cells express serine protease mRNA as a result of systemic mechanisms probably related to inflammation/cytokines before entering inflamed joints.

Salivary levels and immunohistochemical expression of selected angiogenic factors in benign and malignant parotid gland tumours

ObjectivesAngiogenesis underlies tumour growth and metastasis through hepatocyte growth factor (HGF), epithelial growth factor (EGF), and vascular endothelial growth factor (VEGF). The aim of this study was to determine the levels of VEGF, EGF, HGF, HGFR (hepatocyte growth factor receptor), and SRSF1 (serine-rich protein splicing factor-1) in patients with parotid gland tumours and in healthy controls via ELISA in parotid saliva. Immunohistochemical expression of anti-angiogenic isoform of VEGF165b subunit, VEGFR1, VEGFR2, and microvessel density (CD34) were assessed in the tumour tissue and in the non-tumorous surrounding margins.Materials and methodsThe study included 48 patients with benign and malignant parotid gland tumours and 15 healthy controls.ResultsComparison of VEGF, EGF, and HGF in tumour and non-tumorous tissues showed no significant differences and no correlations with tumour stage. The salivary VEGF concentration was significantly higher in patients with pleomorphic adenoma and Warthin’s tumour. No significant correlation was found between expression of VEGF165b and VEGFR2 in tumours and non-tumor surgical margins.ConclusionsThe increased salivary VEGF reflects changes in affected parotid glands, but it cannot be used as a prognostic and differentiative factor for parotid tumours.Clinical relevanceReciprocal relations between growth factors suggest an overlapping pathway of secretion and activity.

Expression of VEGF₁₆₅b, VEGFR1, VEGFR2 and CD34 in benign and malignant tumors of parotid glands

BACKGROUND Vascular endothelial growth factor (VEGF) is an angiogenic factor and could be involved in the pathogenesis of salivary gland tumors. VEGF exerts its biological function by binding to its receptors, VEGFR1 and VEGFR2. An alternative splice variant of VEGF (VEGFxxxb) is an anti-angiogenic factor. Binding VEGF165b with VEGFR2 results in an impaired angiogenic response. The imbalance of VEGFxxx and VEGFxxxb isoforms can underpin pathological angiogenesis. OBJECTIVES The purpose of this study was to evaluate and compare the expression of VEGF165b, VEGFR1, VEGFR2, and CD34 in benign and malignant parotid gland tumors and to explore the possible correlations between their expression and clinicopathological features of tumors. MATERIAL AND METHODS The study was performed on archived paraffin-embedded tissue samples derived from 70 patients with benign and malignant parotid gland tumors (25 with malignant tumors, 23 with pleomorphic adenoma and 22 with Warthins tumor). Immunohistochemical staining of selected tissue sections was performed using monoclonal antibodies. Immunohistochemical staining of selected molecules was used for evaluation of their expression in tissue sections. RESULTS There were no statistically significant differences in the expression of the selected proteins localized in the tumor and surgical margin taken from the same patient. Expression of VEGFR2 correlated with VEGF165b in mixed tumors. There was a statistically significant difference in the expression of VEGFR1 in malignant tumors between females and males, and between the expression of VEGFR1 and the score of T classification in malignant tumors. CONCLUSIONS VEGF165b cannot be treated as a prognostic factor. VEGF receptors correlated with selected clinicopathological data of malignant tumors, indicating their possible role as a prognostic marker. The balance of VEGF isoforms have a limited influence on the development of parotid glands tumors. The correlation between VEGF165b and VEGFR2 in mixed tumors suggests the existence of an additional antiangiogenic pathway in poorly vascularized mixed tumors.

Theeffect of caveolin-1 knockdown on interleukin-1β-inducedchemokine (C-C motif) ligand 2 expression in synovial fluid-derivedfibroblast-like synoviocytes from patients with rheumatoid arthritis

BACKGROUND Rheumatoid arthritis (RA) is a chronic autoimmune disease leading to destructive changes in peripheral joints and their irreversible deformity. The influx of chemoattractant-mediated inflammatory cells to the joints is one of the main features of RA. OBJECTIVES The aim of this study was to investigate the effect of a knockdown of caveolin-1 (CAV1), a known regulator of multiple cell signaling pathways, on chemokine (C-C motif) ligand 2/monocyte chemoattractant protein-1 (CCL2/MCP-1) expression in synovial fluid-derived fibroblast-like synoviocytes (sfd-FLSs) obtained from patients with RA. MATERIAL AND METHODS Primary cell cultures of sfd-FLSs were established from RA synovial fluids. Cells were transiently transfected with small interfering RNA (siRNA) specific for CAV1, and then incubated with interleukin (IL)-1β to induce CCL2 expression. The expression levels of CAV1 and CCL2 were assessed at transcript level, using quantitative polymerase chain reaction (qPCR) and at protein level by enzyme-linked immunosorbent assay (ELISA) and western blotting analysis. RESULTS A transient CAV1 knockdown in sfd-FLSs resulted in a decrease in the IL-1β-induced CCL2 mRNA expression level vs non-transfected cells and cells transfected with non-targeting siRNA. The concentration of secreted CCL2 was not affected significantly. CONCLUSIONS Our study demonstrates that CCL2 expression in sfd-FLSs is CAV1-dependent, but only at transcript level. As the function of CAV1 has not been unequivocally determined, more studies are needed to confirm the role of CAV1 in inflammatory processes related to RA.

Levels of EGF and VEGF in patients with primary and secondary Sjögren’s syndrome

BACKGROUND Aberrant angiogenesis plays a role in the pathogenesis of Sjögrens syndrome (SS). OBJECTIVES The aim of this study was to compare the levels of vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF) in stimulatory parotid saliva and in serum in healthy subjects (HS), patients with primary SS (pSS) and secondary SS (sSS) and to evaluate the expression of EGF, proangiogenic VEGF165 and antiangiogenic VEGF165 b mRNA isoforms. Additionally, we determined the salivary levels of serine/arginine splicing factor (SRSF1), which regulates VEGF165 and VEGF165 b expression. MATERIAL AND METHODS The study comprised 34 women (16 with pSS and 18 with sSS) and healthy subjects for blood and saliva sampling. EGF and VEGF levels in saliva and serum and salivary SRSF1 levels were determined by enzyme-linked immunosorbent assay (ELISA). The expression of VEGF165 , VEGF165 b and EGF in peripheral blood mononuclear cells (PBMC) was evaluated by quantitative polymerase chain reaction (qPCR). RESULTS There were no differences in the levels of EGF, VEGF, SRSF1 and in the expression of the EGF, VEGF165 and VEGF165 b between HS and SS patients, or between pSS and sSS patients. The salivary levels of VEGF165 and EGF were significantly higher in pSS, sSS and HS than serum levels. Levels of SRSF1 correlated positively with VEGF and EGF levels. Levels of EGF, VEGF and SRSF1 correlated with each other. CONCLUSIONS The balance of VEGF isoforms is not disturbed in SS. Saliva is more sensitive for the detection of EGF and VEGF than serum, but salivary levels of those proteins are not representative for SS.

Serum alarm antiproteases in systemic sclerosis patients

Alarm antiproteases, i.e. secretory leukocyte protease inhibitor ad elafin, are key mediators in innate immune response and integrate innate and adaptive immunity systems. The aim of the study was to assess clinical significance of serum levels of alarm antiproteases, elafin and secretory leukocyte protease inhibitor (SLPI) in patients with systemic sclerosis (SSc). Twenty-eight patients with SSc, 25 patients with rheumatoid arthritis (RA) and 22 healthy controls were recruited. Serum elafin and SLPI levels were examined using enzyme-linked immunosorbent assay (ELISA). The patients with SSc had significantly increased serum levels of SLPI in comparison with the RA patients and the healthy controls (p<0.01), and the RA patients presented significantly higher serum levels of elafin in comparison with the controls (p=0.003). In the SSc subgroup serum SLPI level negatively correlated with diffusing capacity of the lung for carbon monoxide (DLCO) (r=-0.41, p=0.03) and total lung capacity (r=-0.42, p=0.03). Both alarm antiproteases, elafin and SLPI could be potentially implicated in the pathogenesis of SSc and SLPI may be considered a candidate for serum biomarker of lung involvement in SSc.

Serum ICAM-1, VCAM-1 and E-selectin levels in patients with primary and secondary Sjögren’s syndrome

BACKGROUND Typical features of Sjögrens syndrome (SS) are severe xerostomia and xerophthalmia which are basic diagnostic criteria. OBJECTIVES The aim of this study was to compare the serum levels of soluble (s) intercellular adhesion molecule 1 (sICAM-1), vascular cell adhesion molecule 1 (sVCAM-1) and sE-selectin between primary (pSS), secondary (sSS) and healthy subjects (HS). We correlated these results with selected clinical parameters of disease activity and parameters of the severity of xerostomia and xerophthalmia. MATERIAL AND METHODS The serum levels of sICAM-1, sVCAM-1 and sE-selectin were determined by enzyme-linked immunosorbent assay (ELISA) in 16 patients with pSS, 18 with sSS and 15 HS. Eye dryness and xerostomia were assessed by the Schirmers test, the Fox test and the visual analogue scale (VAS). RESULTS The levels of sICAM-1 in pSS and sVCAM-1 in sSS patients were significantly higher when compared to HS (p = 0.02 and p = 0.048, respectively). There were no differences between pSS and sSS. In pSS, sVCAM-1 correlated positively with VAS (rS = 0.52, p = 0.04) and the Fox test (rS = 0.66, p=0.01). In sSS, sE-selectin correlated positively with sICAM-1 (rS = 0.54, p = 0.01), the duration of the disease (rS = 0.51, p = 0.03) and negatively with the Schirmers test (rS = 0.59, p = 0.04). sICAM-1 correlated positively with the erythrocyte sedimentation rate (ESR) value (rS = 0.59, p = 0.01). CONCLUSIONS sVCAM-1 reflects xerostomia in pSS. sICAM-1 and sE-selectin may be additional parameters of sSS activity.

Abstract 40: Methylated resveratrol analogue DMU-212 undergoes metabolic activation to DMU-214 in ovarian A-2780 and SKOV-3 cancer cells

Resveratrol (3,49,5-trans-trihydroxystilbene) is a naturally occurring phytoalexin which shows a wide spectrum of biological activity. Although the anticancer effects of resveratrol have been revealed, its clinical application is limited because of a low bioavailability and a rapid clearance from the circulation. The structural alteration of the stilbene motif of resveratrol has been found to be a promising strategy for generation of several synthetic analogues with improved pharmacokinetic parameters. The substitution of hydroxyl groups of resveratrol for methoxy groups may increase the molecule stability, making it less susceptible to phase II conjugation reactions in vivo. The structure-activity studies have revealed that introduction of additional methoxy groups resulted in an increased cytotoxic properties of resveratrol analogues. Furthermore, the methoxy groups at positions 3,5- and 3,4,5- of the trihydroxystilbene scaffold have been identified as crucial for pro-apoptotic activity of the compound. To date few reports have demonstrated the antiproliferative effect of DMU-212 (3,4,49,5-tetramethoxystilbene) in transformed fibroblasts, prostate, liver, colon, cervical, breast and ovarian cancer cells. It has been shown that DMU-212 is biotransformed to four metabolites: 39-hydroxy-3,4,5,49-tetramethoxystilbene (DMU-214), 49-hydroxy-3,4,5-trimethoxystilbene (DMU-281), 4-hydroxy-3,5,49-trimethoxystilbene (DMU-291), and 3-hydroxy-4,5,49-trimethoxystilbene (DMU-807). However, if these metabolites retain the biological activity of DMU-212 remains unknown. Therefore, we evalueted the cytotoxic activity of these four metabolites of DMU-212 and the induction of apoptosis in ovarian A-2780 and SKOV-3 cancer cell lines. CYP1A1 and CYP1B1 are known to catalyze the conversion of estradiol to 4-hydroxyestradiol. Hence, the compounds such as resveratrol or DMU-212, which are structurally similar to the endogenous oestrogen estradiol, have been suggested to undergo metabolism by these enzymes. Surprisingly, DMU-212 has been shown to entirely inhibit CYP1B1 in ovarian A-2780 cancer cell line. Since CYP1B1 has been suggested to be involved in DMU-212 biotransformation, it can be hypothesised that one or more metabolites of DMU-212 regulate the expression of CYP1B1 by the feedback inhibition. To have an insight into DMU-212 metabolites9 role in the expression of isoenzymes of CYP1 family we assessed CYP1A1 and CYP1B1 mRNA and protein levels in metabolites-treated A-2780 and SKOV-3 cancer cells. The inhibitory effect of four metabolites of DMU-212 was investigated in ovarian A-2780 and SKOV-3 cancer cell lines by MTT assays. The induction of apoptosis was assayed by Cell Death Detection ELISAPLUS test, Caspase-Glo® 3/7 luminescent test and flow cytometry analysis. The expression of CYP1A1 and CYP1B1 mRNA and protein levels was investigated by RT-qPCR and Western blott, respectively. From among four metabolites of DMU-212, DMU-214 exhibited the highest cytotoxic activity (IC50 A-2780=13.26±2.11 nM; IC50 SKOV-3=29.21±3.17nM). Furthermore, DMU-214 has been revealed to exert more pronounced inhibitory effects than the prototype compound, DMU-212. In addition, we observed that the stronger cytotoxic activity of DMU-214 evoked its greater pro-apoptotic effect. DMU-214 caused an increase in nucleosomes level in A-2780 and SKOV-3 cells, EF= 15.89 and 7.91, respectively. Exposure of either cell line to DMU-214 resulted in a statistically significant decrease in CYP1A1 and CYP1B1 mRNA and protein levels as compared to control. We showed for the first time that DMU-212 undergoes metabolic activation to DMU-214 in ovarian A-2780 and SKOV-3 cancer cell lines. Since DMU-214 has been observed to decrease expression of CYP1A1 and CYP1B1 in A-2780 and SKOV-3 cells our results provide a new insight into the concept of their feedback inhibition by the compound tested. Citation Format: Sylwia Borys, Malgorzata Kucinska, Dorota Trzybulska, Mariusz Kaczmarek, Marcin Wierzchowski, Marek Murias, Jadwiga Jodynis-Liebert, Hanna Piotrowska. Methylated resveratrol analogue DMU-212 undergoes metabolic activation to DMU-214 in ovarian A-2780 and SKOV-3 cancer cells. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Integrating Clinical Genomics and Cancer Therapy; Jun 13-16, 2015; Salt Lake City, UT. Philadelphia (PA): AACR; Clin Cancer Res 2016;22(1_Suppl):Abstract nr 40.