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For 15 years the agrarian business in our country has been developing in a business environment formed mainly by the common agricultural policy. The purpose of the report is to assess this development in the South-East Planning Region and its impacts on the socio-economic development of rural areas and the state of the environment. The report uses quantitative and qualitative methods. They are the basis of mixed research design. Of the quality methods, expert assessment based on a survey and in-depth structured interviews with 26 experts at the municipal and district level were applied. Quantitative methods are used to process survey and statistical information on agricultural and rural development in the Southeast planning region. The main results are related to the changes in the production and organizational structure of agriculture and their impact on jobs and employment, the age and qualification structure of the employed, unemployment, migration processes, etc. The characteristics of the applied model of agriculture in terms of the predominant type of farms, the functioning of producer organizations, the transition to the production of quality products with protected geographical indications, etc. are outlined.

Aims: Nitric oxide (NO) derived from endothelial NO synthase (eNOS) has been implicated in the adaptive response to hypoxia. An imbalance between 5,6,7,8-tetrahydrobiopterin (BH4) and 7,8-dihydrobiopterin (BH2) can result in eNOS uncoupling and the generation of superoxide instead of NO. Dihydrofolate reductase (DHFR) can recycle BH2 to BH4, leading to eNOS recoupling. However, the role of DHFR and eNOS recoupling in the response to hypoxia is not well understood. We hypothesized that increasing the capacity to recycle BH4 from BH2 would improve NO bioavailability as well as pulmonary vascular remodeling (PVR) and right ventricular hypertrophy (RVH) as indicators of pulmonary hypertension (PH) under hypoxic conditions. Results: In human pulmonary artery endothelial cells and murine pulmonary arteries exposed to hypoxia, eNOS was uncoupled as indicated by reduced superoxide production in the presence of the nitric oxide synthase inhibitor, L-(G)-nitro-L-arginine methyl ester (L-NAME). Concomitantly, NO levels, BH4 availability, and expression of DHFR were diminished under hypoxia. Application of folic acid (FA) restored DHFR levels, NO bioavailability, and BH4 levels under hypoxia. Importantly, FA prevented the development of hypoxia-induced PVR, right ventricular pressure increase, and RVH. Innovation: FA-induced upregulation of DHFR recouples eNOS under hypoxia by improving BH4 recycling, thus preventing hypoxia-induced PH. Conclusion: FA might serve as a novel therapeutic option combating PH. Antioxid. Redox Signal. 23, 1076–1091.

The adenylate cyclase toxin-hemolysin (CyaA, ACT, or AC-Hly) of Bordetella pertussis targets phagocytic cells expressing the complement receptor 3 (CR3, Mac-1, α M β 2 integrin, or CD11b/CD18). CyaA delivers into cells an N-terminal adenylyl cyclase (AC) enzyme domain that is activated by cytosolic calmodulin and catalyzes unregulated conversion of cellular ATP into cyclic AMP (cAMP), a key second messenger subverting bactericidal activities of phagocytes. In parallel, the hemolysin (Hly) moiety of CyaA forms cation-selective hemolytic pores that permeabilize target cell membranes. We constructed the first B. pertussis mutant secreting a CyaA toxin having an intact capacity to deliver the AC enzyme into CD11b-expressing (CD11b + ) host phagocytes but impaired in formation of cell-permeabilizing pores and defective in cAMP elevation in CD11b − cells. The nonhemolytic AC + Hly − bacteria inhibited the antigen-presenting capacities of coincubated mouse dendritic cells in vitro and skewed their Toll-like receptor (TLR)-triggered maturation toward a tolerogenic phenotype. The AC + Hly − mutant also infected mouse lungs as efficiently as the parental AC + Hly + strain. Hence, elevation of cAMP in CD11b − cells and/or the pore-forming capacity of CyaA were not required for infection of mouse airways. The latter activities were, however, involved in bacterial penetration across the epithelial layer, enhanced neutrophil influx into lung parenchyma during sublethal infections, and the exacerbated lung pathology and lethality of B. pertussis infections at higher inoculation doses (>10 7 CFU/mouse). The pore-forming activity of CyaA further synergized with the cAMP-elevating activity in downregulation of major histocompatibility complex class II (MHC-II) molecules on infiltrating myeloid cells, likely contributing to immune subversion of host defenses by the whooping cough agent.

Netherton syndrome (NS) is caused by mutations in the SPINK5 gene. Several Spink5-deficient mouse models were generated to understand the mechanisms of NS in vivo. However, Spink5-deficiency in mice is associated with postnatal lethality that hampers further analysis. Here we present a viable mouse model for NS generated by mosaic inactivation of the Spink5 gene. We propose that these mice are a valuable experimental tool to study NS, especially for long-term studies evaluating potential therapeutic compounds. Furthermore, we show that mosaic inactivation of a gene using TALENs or CRISPR/Cas9 systems can be used to study lethal phenotypes in adult mice.

Matrix metalloproteinases (MMPs) are potential biomarkers for disease activity in inflammatory bowel disease (IBD). However, clinical trials targeting MMPs have not succeeded, likely due to poor understanding of the biological functions of individual MMPs. Here, we explore the role of MMP-19 in IBD pathology. Using a DSS-induced model of colitis, we show evidence for increased susceptibility of Mmp-19-deficient (Mmp-19(-/-)) mice to colitis. Absence of MMP-19 leads to significant disease progression, with reduced survival rates, severe tissue destruction, and elevated levels of pro-inflammatory modulators in the colon and plasma, and failure to resolve inflammation. There was a striking delay in neutrophil infiltration into the colon of Mmp-19(-/-) mice during the acute colitis, leading to persistent inflammation and poor recovery; this was rescued by reconstitution of irradiated Mmp-19(-/-) mice with wild-type bone marrow. Additionally, Mmp-19-deficient macrophages exhibited decreased migration in vivo and in vitro and the mucosal barrier appeared compromised. Finally, chemokine fractalkine (CX3CL1) was identified as a novel substrate of MMP-19, suggesting a link between insufficient processing of CX3CL1 and cell recruitment in the Mmp-19(-/-) mice. MMP-19 proves to be a critical factor in balanced host response to colonic pathogens, and for orchestrating appropriate innate immune response in colitis.

Background Ursodeoxycholic acid (UDCA) is used to treat primary biliary cirrhosis, intrahepatic cholestasis, and other cholestatic conditions. Although much has been learned about the molecular basis of the disease pathophysiology, our understanding of the effects of UDCA remains unclear. Possibly underlying its cytoprotective, anti-apoptotic, anti-oxidative effects, UDCA was reported to regulate the expression of TNFα and other inflammatory cytokines. However, it is not known if this effect involves also modulation of ADAM family of metalloproteinases, which are responsible for release of ectodomains of inflammatory cytokines from the cell surface. We hypothesized that UDCA modulates ADAM17 activity, resulting in amelioration of cholestasis in a murine model of bile duct ligation (BDL). Methods The effect of UDCA on ADAM17 activity was studied using the human liver hepatocellular carcinoma cell line HepG2. Untransfected cells or cells ectopically expressing human ADAM17 were cultured with or without UDCA and further activated using phorbol-12-myristate-13-acetate (PMA). The expression and release of ADAM17 substrates, TNFα, TGFα, and c-Met receptor (or its soluble form, sMet) were evaluated using ELISA and quantitative real-time (qRT) PCR. Immunoblotting analyses were conducted to evaluate expression and activation of ADAM17 as well as the level of ERK1/2 phosphorylation after UDCA treatment. The regulation of tissue inhibitor of metalloproteinases-1 (TIMP-1) by UDCA was studied using zymography and qRT-PCR. A mouse model of acute cholestasis was induced by common BDL technique, during which mice received daily orogastric gavage with either UDCA or vehicle only. Liver injury was quantified using alkaline phosphatase (ALP), relative liver weight, and confirmed by histological analysis. ADAM17 substrates in sera were assessed using a bead multiplex assay. Results UDCA decreases amount of shed TNFα, TGFα, and sMet in cell culture media and the phosphorylation of ERK1/2. These effects are mediated by the reduction of ADAM17 activity in PMA stimulated cells although the expression ADAM17 is not affected. UDCA reduced the level of the mature form of ADAM17. Moreover, UDCA regulates the expression of TIMP-1 and gelatinases activity in PMA stimulated cells. A BDL-induced acute cholangitis model was characterized by increased relative liver weight, serum levels of ALP, sMet, and loss of intracellular glycogen. UDCA administration significantly decreased ALP and sMet levels, and reduced relative liver weight. Furthermore, hepatocytes of UDCA-treated animals retained their metabolic activity as evidenced by the amount of glycogen storage. Conclusions The beneficial effect of UDCA appears to be mediated in part by the inhibition of ADAM17 activation and, thus, the release of TNFα, a strong pro-inflammatory factor. The release of other ADAM17 substrates, TGFα and sMet, are also regulated this way, pointing to a general impact on the release of ADAM17 substrates, which are pivotal for liver regeneration and function. In parallel, UDCA upregulates TIMP-1 that in turn inhibits matrix metalloproteinases, which destroy the hepatic ECM in diseased liver. This control of extracellular matrix turnover represents an additional beneficial path of UDCA treatment.

Patients with hormone refractory prostate cancer (HRPC) have very few clinical options. A bottle neck in development of new therapies is the poor understanding of the molecular basis of hormone independency. A pathway thought to be important in this progression is FGFR. Amplification of FGFR1 has been observed in breast cancer and has led to the hypothesis that it is involved in Tamoxifen resistance (Turner et al., 2010). Here we evaluated the copy number variations (CNV) of the gene loci of FGFR1 (8p12) and FGFR2 (10q26) by dual color FISH in FFPE tissue of recurrent PC patients (n=28), non-recurrent PC patients (n=15), PC patients undergoing palliative RPE (n=15), patients with benign prostate hyperplasia (n=17) and tumor free tissue samples (n=12). We analyzed CNV between different clinical patient subgroups. Two types of CNV for the gene loci of FGFR1 and FGFR2 were observed. Either the gene dose of the target gene locus occurred to be amplified or the gene dose was elevated due to polysomy of the respective chromosome. Furthermore patients with CNV of FGFR1 or FGFR2 exhibited in 40-50% intra tumor heterogeneity by presenting normal cells and cells with CNV. FGFR1 gene dose elevations (either due to gene locus amplification or polysomy of chromosome 8) were observed in 7% (1/15) of patients without recurrence but in 49% (17/35) of the patients with recurrence. Also, in patients undergoing palliative RPE 75% (12/16) of the patients exhibited FGFR1 gene dose elevation. In the case of FGFR2, gene dose elevations (gene locus amplification or polysomy of chromosome 10) were observed in none of the patients without recurrence but in 13% of the patients with recurrence and in 20% of patients undergoing palliative RPE. In the control groups such variations were not found. Interestingly, the gene dose variations differed in the prostate cancer patients depending on their hormone sensitivity. 47% (7/15) of hormone refractory patients and 30% (6/20) of the hormone sensitive patients showed elevated FGFR1 gene dose, for FGFR2, 30% and 20% of the respective subgroups were affected. In summary we conclude that this is first evidence that CNV of FGFR1 and FGFR2 might have implications for hormone resistance in PC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3211. doi:10.1158/1538-7445.AM2011-3211