Despite the importance of circulating microparticles in haemostasis and thrombosis, there is limited evidence for potential causative effects of naturally produced cell-derived microparticles on fibrin clot formation and its properties. We studied the significance of blood microparticles for fibrin formation, structure and susceptibility to fibrinolysis by removing them from platelet-free plasma using filtration. Clots made in platelet-free and microparticle-depleted plasma samples from the same healthy donors were analyzed in parallel. Microparticles accelerate fibrin polymerisation and support formation of more compact clots that resist internal and external fibrinolysis. These variations correlate with faster thrombin generation, suggesting thrombin-mediated kinetic effects of microparticles on fibrin formation, structure and properties. In addition, clots formed in the presence of microparticles, unlike clots from the microparticle-depleted plasma, contain 0.1–0.5-μm size granular and CD61-positive material on fibres, suggesting that platelet-derived microparticles attach to fibrin. Therefore, the blood of healthy individuals contains functional microparticles at the levels that have a procoagulant potential. They affect the structure and stability of fibrin clots indirectly through acceleration of thrombin generation and through direct physical incorporation into the fibrin network. Both mechanisms underlie a potential role of microparticles in haemostasis and thrombosis as modulators of fibrin formation, structure and resistance to fibrinolysis.

The microtubule-targeting agents (MTAs) are well-known chemotherapeutic agents commonly used for therapy of a broad spectrum of human malignancies, exhibiting epithelial origin, including breast, lung, and prostate cancer. Despite the impressive response rates shortly after initiation of MTA-based therapy, the vast majority of human malignancies develop resistance to MTAs due to the different mechanisms. Here, we report that infigratinib (BGJ 398), a potent FGFR1-4 inhibitor, restores sensitivity of a broad spectrum of ABCB1-overexpressing cancer cells to certain chemotherapeutic agents, including paclitaxel (PTX) and doxorubicin (Dox). This was evidenced for the triple-negative breast cancer (TNBC), and gastrointestinal stromal tumor (GIST) cell lines, as well. Indeed, when MDR-overexpressing cancer cells were treated with a combination of BGJ 398 and PTX (or Dox), we observed a significant increase of apoptosis which was evidenced by an increased expression of cleaved forms of PARP, caspase-3, and increased numbers of Annexin V-positive cells, as well. Moreover, BGJ 398 used in combination with PTX significantly decreased the viability and proliferation of the resistant cancer cells. As expected, no apoptosis was found in ABCB1-overexpressing cancer cells treated with PTX, Dox, or BGJ 398 alone. Inhibition of FGFR-signaling by BGJ 398 was evidenced by the decreased expression of phosphorylated (i.e., activated) forms of FGFR and FRS-2, a well-known adaptor protein of FGFR signaling, and downstream signaling molecules (e.g., STAT-1, -3, and S6). In contrast, expression of MDR-related ABC-transporters did not change after BGJ 398 treatment, thereby suggesting an impaired function of MDR-related ABC-transporters. By using the fluorescent-labeled chemotherapeutic agent PTX-Alexa488 (Flutax-2) and doxorubicin, exhibiting an intrinsic fluorescence, we found that BGJ 398 substantially impairs their efflux from MDR-overexpressing TNBC cells. Moreover, the efflux of Calcein AM, a well-known substrate for ABCB1, was also significantly impaired in BGJ 398-treated cancer cells, thereby suggesting the ABCB1 as a novel molecular target for BGJ 398. Of note, PD 173074, a potent FGFR1 and VEGFR2 inhibitor failed to retain chemotherapeutic agents inside ABCB1-overexpressing cells. This was consistent with the inability of PD 173074 to sensitize Tx-R cancer cells to PTX and Dox. Collectively, we show here for the first time that BGJ 398 reverses the sensitivity of MDR-overexpressing cancer cells to certain chemotherapeutic agents due to inhibition of their efflux from cancer cells via ABCB1-mediated mechanism.

Deregulation of receptor tyrosine kinase (RTK)-signaling is frequently observed in many human malignancies, making activated RTKs the promising therapeutic targets. In particular, activated RTK-signaling has a strong impact on tumor resistance to various DNA damaging agents, e.g., ionizing radiation and chemotherapeutic drugs. We showed recently that fibroblast growth factor receptor (FGFR)-signaling might be hyperactivated in imatinib (IM)-resistant gastrointestinal stromal tumors (GIST) and inhibition of this pathway sensitized tumor cells to the low doses of chemotherapeutic agents, such as topoisomerase II inhibitors. Here, we report that inhibition of FGFR-signaling in GISTs attenuates the repair of DNA double-strand breaks (DSBs), which was evidenced by the delay in γ-H2AX decline after doxorubicin (Dox)-induced DNA damage. A single-cell gel electrophoresis (Comet assay) data showed an increase of tail moment in Dox-treated GIST cells cultured in presence of BGJ398, a selective FGFR1-4 inhibitor, thereby revealing the attenuated DNA repair. By utilizing GFP-based reporter constructs to assess the efficiency of DSBs repair via homologous recombination (HR) and non-homologous end-joining (NHEJ), we found for the first time that FGFR inhibition in GISTs attenuated the homology-mediated DNA repair. Of note, FGFR inhibition/depletion did not reduce the number of BrdU and phospho-RPA foci in Dox-treated cells, suggesting that inhibition of FGFR-signaling has no impact on the processing of DSBs. In contrast, the number of Dox-induced Rad51 foci were decreased when FGFR2-mediated signaling was interrupted/inhibited by siRNA FGFR2 or BGJ398. Moreover, Rad51 and -H2AX foci were mislocalized in FGFR-inhibited GIST and the amount of Rad51 was substantially decreased in -H2AX-immunoprecipitated complexes, thereby illustrating the defect of Rad51 recombinase loading to the Dox-induced DSBs. Finally, as a result of the impaired homology-mediated DNA repair, the increased numbers of hypodiploid (i.e., apoptotic) cells were observed in FGFR2-inhibited GISTs after Dox treatment. Collectively, our data illustrates for the first time that inhibition of FGF-signaling in IM-resistant GIST interferes with the efficiency of DDR signaling and attenuates the homology-mediated DNA repair, thus providing the molecular mechanism of GIST’s sensitization to DNA damaging agents, e.g., DNA-topoisomerase II inhibitors.

Allergic rhinitis (AR) is especially prevalent among the population of large cities. Immunologically, the airway epithelium is a region where the population of allergen-presenting cells concentrates. These cells actively express a group of receptors of the innate immune system. A specific cytokine profile is its representation. The study was aimed at evaluating the cytokine profile in patients with seasonal and perennial allergic rhinitis. The cytokine profile of nasal secretion and blood serum of 44 patients with AR was studied. 24 of them had seasonal allergic rhinitis (SAR), and 20 patients suffered from perennial allergic rhinitis (PAR). The patients’ age ranged from 4 to 60 years. It was determined in our study that the activation of the GM-CSF production retained in patients with PAR sensitized to mite allergen components (Dermatophagoides pteronyssinus). There was a higher production profile of TNF-α and TSLP in nasal secretion in the patients with perennial allergic rhinitis and additional high sensitization to SEs. Sensitization to mold fungal allergen components significantly increases in patients with seasonal allergic rhinitis. They demonstrated high level of sensitization to the Aspergillus fumigatus component m3. Thus, along with other clinical trials, the study performed would clarify some aspects of molecular pathogenesis of human allergic rhinitis.

The acquired resistance of gastrointestinal stromal tumors (GISTs) to the targeted-based therapy remains the driving force to identify the novel approaches that are capable of increasing the sensitivity of GISTs to the current therapeutic regimens. Our present data show that BGJ398, a selective fibroblast growth factor receptor (FGFR) inhibitor, sensitizes imatinib (IM)-resistant GIST cells with receptor tyrosine kinase (RTK) switch (loss of c-KIT/gain of pFGFR2a) to the low doses of topoisomerase II inhibitors – doxorubicin (Dox) and etoposide (Eto). Mechanistically, pretreatment of IM-resistant GIST cells with BGJ398 for 12 h markedly enhanced proapoptotic and growth-suppressive effects of Dox (or Eto). Indeed, a significant cleavage of PARP and caspase-3 was observed in GIST cells treated with a combination of FGFR and topoisomerase II inhibitor. In contrast, no signs of apoptosis were detected in IM-resistant GIST cells treated with BGJ398, whereas the low doses of Dox (Eto) exerted the minor proapoptotic effects on GISTs. The mechanism of BGJ398-induced sensitization of GIST to topoisomerase II inhibitors might be because of attenuation of DNA damage signaling and repair. Indeed, we observed a marked decrease in Rad51 expression in GIST cells treated with BGJ398 together with Dox. Similar results were obtained when an overexpressed pFGFR2a was knocked down by corresponding siRNA before Dox (Eto) exposure. Moreover, FGFR inhibition/depletion caused a loss of Rad51 foci in Dox-treated GIST cells, suggesting that FGFR-signaling plays an important regulatory role in homology-mediated DNA repair. Our data show that combined therapy (RTKs inhibitors supplemented with low doses of topoisomerase II inhibitors) might be effective for unresectable and metastatic forms of GISTs. In case of resistance to IM because of RTKs switch indicated above, FGFR inhibitors (e.g. BGJ398) might be potentially useful because of their ability to sensitize tumor cells to topoisomerase II inhibitors and induce tumor cell apoptosis by targeting DNA double-strand breaks repair.