Prognostic significance of Dicer cellular levels in soft tissue sarcomas

In the present study we assessed the expression and distribution of endoribonuclease Dicer in soft tissue tumors and correlated its cellular levels with clinicopathological parameters, including clinical outcome. Dicer was expressed in the tested cell line as well as in the majority of the sarcomas examined. Staining intensity was significantly higher in sarcomas compared with benign neoplasms and in high-grade compared with low-grade tumors. Elevated Dicer immunoreactivity was strongly associated with poor outcome and Dicer cellular levels were an independent negative prognostic factor.     

Characteristics of the minor salivary gland infiltrates in Sjögren's syndrome

Sjögren's syndrome (SS) is a chronic autoimmune exocrinopathy associated with variable degree of lymphocytic infiltration of the affected organs (primarily salivary and lacrimal glands) and broad clinical manifestations. Minor salivary gland (MSG) lesions mainly consist of T and B cells, while antigen-presenting cells have been reported in heavy infiltrates. Evidence suggests that the infiltrate composition differs according to lesion severity; however, these differences are not well-defined. To investigate the differential distribution of the major infiltrating mononuclear cell (MNC) types in SS-lesions of variable severity, total-T, CD4⁺-T, CD8⁺-T, Treg, and B cell, macrophage (MΦ), interdigitating (iDC) and follicular dendritic cell (fDC), and natural-killer (NK)-cell incidence (%-total infiltrating MNC) was analyzed in MSG biopsies with mild (n = 11), intermediate (n = 13) or severe (n = 15) lesions. T cells, CD4⁺-T cells and Tregs, B lymphocytes, MΦs and iDCs were significantly different among MSG tissues with mild, intermediate or severe inflammatory lesions, while CD8⁺-T cell, fDC and NK cell incidence was not correlated with lesion severity. T cell, CD4⁺-T cell, T/B cell ratio and iDC incidence was negatively, whereas B cell and MΦ incidence was positively correlated with infiltration grade and biopsy focus score. Tregs predominated in intermediate lesions. Multivariate analysis revealed several associations between the incidence of each infiltrating MNC-type and disease manifestations, implying an involvement of local immune responses in systemic disease features. Our findings support that the distribution of infiltrating MNCs at the SS-lesions varies according to lesion severity and correlates with disease manifestations. The significance of this differential distribution and the underlying aetiopathogenic factors need to be elucidated.     

Precise probes of type II interferon activity define the origin of interferon signatures in target tissues in rheumatic diseases

Elucidating the molecular pathways active in pathologic tissues has important implications for defining disease subsets, selecting therapy, and monitoring disease activity. The development of therapeutics directed at IFN-α or IFN-γ makes the discovery of probes that report precisely on the activity of different IFN pathways a high priority. We show that, although type I and II IFNs induce the expression of a largely overlapping group of molecules, precise probes of IFN-γ activity can be defined. Used in combination, these probes show prominent IFN-γ effects in Sjögren syndrome (SS) tissues. In contrast, dermatomyositis muscle shows a dominant type I IFN pattern. Interestingly, heterogeneity of IFN signatures exists in patients with SS, with some patients demonstrating a predominant type I pattern. The biochemical patterns largely distinguish the target tissues in patients with SS from those with dermatomyositis and provide a relative weighting of the effects of distinct IFN pathways in specific biopsies. In SS, type I and II IFN effects are localized to the same epithelial cells, surrounded by inflammatory cells expressing IFN-γ–induced proteins, suggesting reinforcing interactions. Precise probes of the different IFN pathways active in tissues of complex rheumatic diseases will be critical to classify disease, elucidate pathogenesis, and select therapy.     

The combination of antitumor drugs,exemestane and erlotinib,induced resistance mechanism in H358 and A549 non-small cell lung cancer (NSCLC) cell lines

Context: Estrogens in non-small-cell lung cancer (NSCLC) are important, and their interaction with epidermal growth factor receptor (EGFR) might be crucial.

Objective: This study investigates the effect of exemestane, an aromatase inhibitor, and erlotinib, an EGFR inhibitor, on human NSCLC cell lines; H23, H358 and A549.

Materials and methods: A cell proliferation assay was used for measuring cell number, apoptosis assay for detecting apoptosis and necrosis and immunoblotting for beclin-1 and Bcl-2 proteins detection. An immunofluorescence assay was used for EGFR localization. A migration assay and zymography were used for cell motility and metalloproteinases (MMPs) expression, respectively.

Results: Exemestane, erlotinib or their combination decreased cell proliferation and increased apoptosis. Exemestane’s half maximal inhibitory concentration (IC₅₀) was 50?μM for H23 and H358 cells and 20?μM for A549. The IC₅₀ of erlotinib was 25?μM for all cell lines. Apoptosis increase induced by exemestane was 58.0 (H23), 186.3 (H358) and 34.7% (A549) and by erlotinib was 16.7 (H23), 65.3 (H358) and 66.3% (A549). A synergy effect was observed only in H23 cells. Noteworthy, the combination of exemestane and erlotinib decreased beclin-1 protein levels (32.3?±?19.2%), an indicator of autophagy, in H23 cells. The combination of exemestane and erlotinib partially reversed the EGFR translocation to mitochondria and decreased MMP levels and migration.

Discussion and conclusions: The benefit from a dual targeting of aromatase and EGFR seems to be regulated by NSCLC cell content. The diverse responses of cells to agents might be influenced by the dominance of certain molecular pathways.     

Oxidative stress due to radiation in CD34+ Hematopoietic progenitor cells: protection by IGF-1

Radiation exerts direct as well as indirect effects on DNA through the generation of reactive oxygen species (ROS). Irradiated hematopoietic progenitor cells (HPCs) experience DNA strand breaks, favoring genetic instability, due to ROS generation. Our aim was to study the effect of a range of radiation doses in HPCs and the possible protective mechanisms activated by insulin-like growth factor-1 (IGF-1). ROS generation was evaluated, in the presence or absence of IGF-1 in liquid cultures of human HPCs-CD34⁺ irradiated with 1-, 2- and 5-Gy X-rays, using a flow cytometry assay. Manganese superoxide dismutase (MnSOD) expression was studied by western blot analysis and visualized by an immunofluorescence assay. Apoptosis was estimated using the following assays: Annexin-V assay, DNA degradation assay, BCL-2/BAX mRNA and protein levels and caspase-9 protein immunofluorescence visualization. Viability and clonogenic potential were studied in irradiated HPCs. The generation of superoxide anion radicals at an early and a late time point was increased, while the hydrogen peroxide generation at a late time point was stable. IGF-1 presence further enhanced the radiation-induced increase of MnSOD at 24 h post irradiation. IGF-1 inhibited the mitochondria-mediated pathway of apoptosis by regulating the m-RNA and protein expression of BAX, BCL-2 and the BCL-2/BAX ratio and by decreasing caspase-9 protein expression. IGF-1 presence in culture media of irradiated cells restored the clonogenic capacity and the viability of HPCs as well. In conclusion, IGF-1 protects HPCs-CD34⁺ from radiation effects, by eliminating the oxidative microenvironment through the enhancement of MnSOD activation and by regulating the mitochondria-mediated pathway of apoptosis.     

Association of monocyte chemoattractant protein-1 −2518A>G polymorphism with occurrence, severity, and outcome in ischemic stroke

Monocyte chemoattractant protein-1 (MCP-1) is implicated in promoting atherosclerotic diseases, including stroke. Therefore, several studies have investigated the association between variants of the MCP-1 gene and risk of atherosclerotic diseases. We sought to determine the occurrence of MCP-1 ?2518A>G polymorphism in patients with ischemic stroke (IS), and studied its association with the severity of disease and functional outcome after an acute IS. One hundred and forty-five consecutive patients with first ever IS and 145 age- and sex-matched control subjects were recruited. Stroke severity and functional outcome were assessed on admission and at one month post-stroke, respectively. Genotyping for the MCP-1 ?2518A>G polymorphism was performed by a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). No significant difference in the frequency of MCP-1 ?2518A>G genotypes between IS patients and controls was found, with OR = 0.69 (95 % CI 0.46–1.04, P = 0.08). Moreover, carriage of the G allele was not associated with stroke severity (Scandinavian stroke scale score 33.1 vs. 32.5, respectively, P = 0.71), or poor outcome at 1 month post-stroke (63.9 vs. 59.7 %, respectively, P = 0.61). In conclusion, we were unable to demonstrate a significant association of the MCP-1 ?2518A>G gene polymorphism with IS occurrence, severity or functional outcome in a Caucasian population. However, larger studies are necessary to fully elucidate the role of this polymorphism in IS.     

Functional estrogen receptors alpha and beta are expressed in normal human salivary gland epithelium and apparently mediate immunomodulatory effects

Salivary gland epithelial cells (SGECs) have been shown to participate in immunological responses and have been implicated in the pathogenesis of Sjögren's syndrome (SS). Experimental evidence from animal models indicates that estrogen deficiency may also participate in SS pathogenesis. However, the expression and functionality of the estrogen receptors alpha (ERα) and beta (ERβ) in normal human salivary epithelium is unknown. To investigate these points, formalin-fixed, paraffin-embedded specimens and cultured non-neoplastic SGEC lines derived from nine minor salivary gland (MSG) biopsies with normal histology were studied. Immunohistochemical analyses detected the epithelial expression of ERα, ERβ1, and ERβ2 protein isoforms both in MSG tissues and in cultured SGECs. Such epithelial expression was verified by immunoblotting of various ER proteins in cellular extracts of cultured SGECs (full-length-ERα, ERα-Δ3, ERβ1-long, ERβ1-short, and ERβ2-long isoforms). Estrogens did not induce growth or apoptosis in cultured SGECs. However, similarly to other cellular systems, treatment of cultured SGECs with estrogens (17β-estradiol and the ERα- and ERβ-selective agonists propylpyrazole-triol and diarylpropiolnitrile, respectively) inhibited the interferon-γ-inducible expression of intercellular adhesion molecule-1. This finding corroborated the functionality of ER expressed by SGEC. Our results suggest that salivary epithelium expresses constitutively functional ERα and ERβ proteins that apparently mediate immunomodulatory effects.     

Oval cell proliferation in cirrhosis in rats. An experimental study

Aim: Oval cells are liver stem cells involved in liver regeneration following liver damage. Previous studies have shown that pretreatment with a hepatocyte inhibitor is required to allow full oval cell activation. This study investigates whether oval cells develop and proliferate in a model of experimental liver fibrosis without pretreatment with a known hepatocyte inhibitor. Methods: The study comprised 66 male Wistar rats divided into two groups: A (n = 6): controls; and B (n = 60): CCl(4) injection (intraperitoneally 2 mL/kg bodyweight 1:1 volume in corn oil twice weekly). Rats were sacrificed at four, eight and 12 weeks. Liver tissues were evaluated for the degree of fibrosis (Masson's trichrome), cell proliferation (Ki67 antigen), expression of alpha-fetoprotein (AFP) mRNA (RT-PCR and in situ hybridization), AFP protein (Western blot) and cytokeratin-19. Cells with morphologic features of oval cells that were cytokeratin 19 (CK19)+ and AFP mRNA+ were scored in morphometric analysis. Results: Oval cells were present in all 66 specimens; their percentage was higher in group B compared to group A (P < 0.001). AFP mRNA and protein expression increased as fibrosis advanced. Similarly, the numbers of CK19+, AFP mRNA+ and Ki67+ oval cells were higher in advanced fibrosis stages. Conclusion: This study demonstrates that oval cells develop and proliferate in a model of experimental liver fibrosis without pretreatment with a known hepatocytic inhibitor. However, further research is warranted in order to identify the exact molecular mechanisms involved in this process.     

Conceptual Model for Heart Failure Disease Management

The objective of this review is to propose a conceptual model for heart failure (HF) disease management (HFDM) and to define the components of an efficient HFDM plan in reference to this model. Articles that evaluated 1 or more of the following aspects of HFDM were reviewed: (1) outpatient clinic follow-up; (2) self-care interventions to enhance patient skills; and (3) remote evaluation of worsening HF either using structured telephone support (STS) or by monitoring device data (telemonitoring). The success of programs in reducing readmissions and mortality were mixed. Outpatient follow-up programs generally resulted in improved outcomes, including decreased readmissions. Based on 1 meta-analysis, specialty clinics improved outcomes and nonspecialty clinics did not. Results from self-care programs were inconsistent and might have been affected by patient cognitive status and educational level, and intervention intensity. Telemonitoring, despite initially promising meta-analyses demonstrating a decrease in the number and duration of HF-related readmissions and all-cause mortality rates at follow-up, has not been shown in randomized trials to consistently reduce readmissions or mortality. However, evidence from device monitoring trials in particular might have been influenced by technology and design issues that might be rectified in future trials. Results from the literature suggest that the ideal HFDM plan would include outpatient follow-up at an HF specialty clinic and continuous education to improve patient self-care. The end result of this plan would lead to better understanding on the part of the patient and improved patient ability to recognize and respond to signs of decompensation.     

An in vitro study,evaluating the effect of sunitinib and/or lapatinib on two glioma cell lines

Aim We sought to assess the effect of sunitinib and lapatinib applied either alone or in combination, on U87 and M059K glioma cells. Methods Both cell lines were cultured as recommended by the manufacturer. Sunitinib and lapatinib were applied, either separately or in combination, in the cultured cells after cell attachment at doses of 10nM, 100nM, 1 μM and 10 μM. To determine whether the agents affect the proliferation of glioma cells, the 3-[4,5-dimethylthiazol-2-yl]-2,5 dimethyltetrazolium bromide assay was used. Apoptosis was detected using annexin V/propidium iodide detection assay, migration assay was performed in 24-well microchemotaxis chambers. The release of MMPs into the culture medium of U87 and M059K cells was measured by zymography. Results Both agents, administered either alone or in combination, decreased cell proliferation in a dose-dependent manner 48 h after their application in both cell lines. The inhibition of their combination was statistically different than the inhibition of each agent alone. Apoptosis was increased and migration of U87 and M059K cells was inhibited either by each agent alone or their combination. MMPs levels remained unaffected by the application of both agents in U87 cells. However, MMP-9 and MMP-2 levels were decreased 48 h after treatment of M059K cells with sunitinib either alone or in combination with lapatinib. Conclusion Sunitinib and/or lapatinib appear to exhibit significant effects on proliferation, apoptosis and migration of glioma cells. When applied alone, sunitinib appears to be a more potent inhibitor than lapatinib.