Increased production of inflammatory cytokines by circulating monocytes in mesial temporal lobe epilepsy: A possible role in drug resistance.

We analyzed peripheral blood mononuclear cells (PBMCs) and serum inflammatory biomarkers in patients with mesial temporal lobe epilepsy (drug-resistant - DR, vs. drug-sensitive - DS). Patients with epilepsy showed higher levels of serum CCL2, CCL3, IL-8 and AOPP, and lower levels of FRAP and thiols compared to healthy controls (HC). Although none of the serum biomarkers distinguished DR from DS patients, when analysing intracellular cytokines after in vitro stimulation, DR patients presented higher percentages of IL-1ß and IL-6 positive monocytes compared to DS patients and HC. Circulating innate immune cells might be implicated in DR epilepsy and constitute potential new targets for treatments.

Mesangiogenic Progenitor Cells and musculoskeletal tissue regeneration: differences between adipose-derived and bone marrow-derived cells?

Mesangiogenic Progenitor cells (MPCs) have been isolated from human bone marrow mononuclear cells (hBM-MNCs) and attracted particular attention for their ability to efficiently differentiate into exponentially growing mesenchymal stromal cells (MSCs) and toward endothelial lineage, suggesting the term "mesangiogenic". Coupling mesengenesis and angiogenis, MPCs has been hypothesized retaining a great tissue regenerative potential in musculoskeletal tissues regeneration. Bone marrow and adipose tissue (AT) represent most promising adult multipotent cell sources attempting to repair bone and cartilage, with controversial results regarding advantages applying BM- or AT-derived cells. As different culture determinants as well as tissue of origins, could strongly affect regenerative potential of cell preparations, we hypothesize that MPCs counterpart could have a role in defining efficacy of applying a cell-based medicinal product in musculoskeletal tissue repair. Here we present convincing data demonstrating that the ex vivo progenitors of MPCs are tissue specific and can be detected exclusively in hBM-MNCs.

Embedding methods for poly(L-lactic acid) microfiber mesh/human mesenchymal stem cell constructs.

Fiber mesh scaffolds were recently investigated in tissue engineering as possible support for stem cell growth and differentiation, in order to repair lesion areas in clinical practice. In particular, the literature is focused on fiber mesh scaffolds constituted of biocompatible and resorbable polymeric structures, like poly(L-lactic acid) (PLLA). However, as regards the study of constructs constituted of PLLA microfibers and cells, only quantitative and SEM analyses were reported, lacking histological analysis. Histological evaluation of these constructs could give important information about cellular distribution in the scaffold, cell-scaffold interactions and extracellular matrix production. The purpose of our study was to find a valid method to analyze PLLA microfiber/cell constructs from both histological and histochemical angles. Biodegradable non-woven fiber meshes were prepared using hollow microfibers, based on PLLA. We first evaluated different embedding methods useable for histological analysis and the results showed that among the paraffin, Killik, and acrylic resin the only suitable medium was the latter. Then we employed the acrylic resin to embed the constructs made up of PLLA microfibers and bone marrow-derived human mesenchymal stromal cells, which we then analyzed with Toluidine Blue, PAS and Alcian Blue staining. These constructs, previously analyzed for cell viability by MTT and CCK-8 tests, showed viable/proliferating cells until 6 weeks of culture. The stainings performed on constructs confirmed viability data obtained with SEM and MTT/CCK-8 and supplied other information on the cell behaviors such as the distribution and organization onto the scaffold and the production of extracellular matrix molecules. In conclusion, this methodological study mainly suggests a suitable method to analyze PLLA microfiber/cell constructs, at the same time confirming and enriching the literature data on the compatibility between PLLA microfibers and hMSCs.

Interaction between gemcitabine and topotecan in human non-small-cell lung cancer cells: effects on cell survival, cell cycle and pharmacogenetic profile.

The pyrimidine analogue gemcitabine is an established effective agent in the treatment of non-small-cell lung cancer (NSCLC). The present study investigates whether gemcitabine would be synergistic with the topoisomerase I inhibitor topotecan against the NSCLC A549 and Calu-6 cells. Cells were treated with gemcitabine and topotecan for 1 h and the type of drug interaction was assessed using the combination index (CI). Cell cycle alterations were analysed by flow cytometry, while apoptosis was examined by the occurrence of DNA internucleosomal fragmentation, nuclear condensation and caspase-3 activation. Moreover, the possible involvement of the PI3K-Akt signalling pathway was investigated by the measurement of Akt phosphorylation. Finally, quantitative, real-time PCR (QRT-PCR) was used to study modulation of the gemcitabine-activating enzyme deoxycytidine kinase (dCK) and the cellular target enzyme ribonucleotide reductase (RR). In results, it was found that simultaneous and sequential topotecan --> gemcitabine treatments were synergistic, while the reverse sequence was antagonistic in both cell lines. DNA fragmentation, nuclear condensation and enhanced caspase-3 activity demonstrated that the drug combination markedly increased apoptosis in comparison with either single agent, while cell cycle analysis showed that topotecan increased cells in S phase. Furthermore, topotecan treatment significantly decreased the amount of the activated form of Akt, and enhanced the expression of dCK (+155.0 and +115.3% in A549 and Calu-6 cells, respectively), potentially facilitating gemcitabine activity. In conclusion, these results indicate that the combination of gemcitabine and topotecan displays schedule-dependent activity in vitro against NSCLC cells. The gemcitabine --> topotecan sequence is antagonistic while drug synergism is obtained with the simultaneous and the sequential topotecan --> gemcitabine combinations, which are associated with induction of decreased Akt phosphorylation and increased dCK expression.

Variable correlation between 6-mercaptopurine metabolites in erythrocytes and hematologic toxicity: implications for drug monitoring in children with acute lymphoblastic leukemia.

Nineteen pediatric patients affected by acute lymphoblastic leukemia (ALL) were examined weekly with respect to 6-mercaptopurine nucleotide (6-MPN) and 6-thioguanine nucleotide (6-TGN) levels in erythrocytes during the course of maintenance treatment with 6-MP 50 mg/m2 per d and results were related to various parameters of bone marrow function to assess, in the same individual, the level of reliability of 6-MP metabolites in predicting a later change in peripheral blood cell counts. Median values for 6-MPN and 6-TGN were 57 and 200 pmol/8 x 10(8) erythrocytes, respectively, as measured by reversed-phase high-performance liquid chromatography (HPLC). 6-TGN levels in erythrocytes were inversely related with white blood cell count (r = -0.463, p < 0.0001, n = 361), absolute neutrophil count (r = -0.386, p < 0.0001, n = 347), erythrocyte (r = -0.354, p < 0.0001, n = 287), and platelet counts (r = -0.24, p < 0.0001, n = 319) in the majority of patients (n = 10-12), while no correlation was found for 6-MPN. In the remaining children, no evidence of correlation was demonstrated between 6-TGN levels and myelotoxicity. The results confirm the role of 6-TGN as the reference cytotoxic metabolite for evaluating the exposure to 6-MP and identifying treatment compliance in ALL children but indicate the limits of a follow-up based solely on metabolite levels and suggest that a more correct approach remains the double monitoring of 6-TGN and blood cell count with differential.

Manumycin inhibits ras signal transduction pathway and induces apoptosis in COLO320-DM human colon tumour cells.

The aim of the present study was to assess the cytotoxicity of manumycin, a specific inhibitor of farnesyl:protein transferase, as well as its effects on protein isoprenylation and kinase-dependent signal transduction in COLO320-DM human colon adenocarcinoma which harbours a wild-type K-ras gene. Immunoblot analysis of isolated cell membranes and total cellular lysates of COLO320-DM cells demonstrated that manumycin dose-dependently reduced p21 ras farnesylation with a 50% inhibitory concentration (IC50) of 2.51 +/- 0.11 microM and 2.68 +/- 0.20 microM, respectively, while the geranylgeranylation of p21 rhoA and p21rap1 was not affected. Manumycin dose-dependently inhibited (IC50 = 2.40 +/- 0.67 microM) the phosphorylation of the mitogen-activated protein kinase/extracellular-regulated kinase 2 (p42MAPK/ERK2), the main cytoplasmic effector of p21ras, as well as COLO320-DM cell growth (IC50 = 3.58 +/- 0.27 microM) without affecting the biosynthesis of cholesterol. Mevalonic acid (MVA, 100 microM), a substrate of the isoprenoid synthesis, was unable to protect COLO320-DM cells from manumycin cytotoxicity. Finally, manumycin 1-25 microM for 24-72 h induced oligonucleosomal fragmentation in a dose- and time-dependent manner and MVA did not protect COLO320-DM cells from undergoing DNA cleavage. The present findings indicate that the inhibition of p21ras processing and signal transduction by manumycin is associated with marked inhibition of cell proliferation and apoptosis in colon cancer cells and the effect on cell growth does not require the presence of a mutated ras gene for maximal expression of chemotherapeutic activity.