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Clusterin/Apolipoprotein J (CLU) is a cellular senescence biomarker implicated in several physiological processes. In this work we have investigated CLU expression and function in human haematopoietic cells. We found that early passage human T cell clones (TCC) express minimal endogenous amounts of CLU, which are significantly elevated in late passage cells. Moreover, exposure of TCC to increased levels of the essential micronutrient zinc in culture resulted in intense induction of CLU. Because haematopoietic cells cease proliferation following induction of terminal differentiation, we also studied the expression profile of CLU in the leukemic progenitor cell lines K562 and HL-60. We found that, like TCC, both cell lines express minimal endogenous levels of CLU in their actively proliferating state. However, when induced to differentiate into their distinct cell types, CLU was found to be up-regulated specifically in those cells expressing the main differentiation markers. Enforced stable over-expression of CLU in K562 cells inhibited the expression of the CD14 differentiation marker and blocked differentiation to either monocytes/megacaryoblasts or to erythrocytes. Overall, our results suggest that CLU is actively involved in both replicative senescence and terminal differentiation in different types of human haematopoietic cells.

Immunoporation has been found to be able to efficiently transfect a wide range of human cultured cell lines. This report shows that peripheral blood mononuclear cells can also be efficiently transfected using immunoporation. The immunoporation of the cells with fluorescent TMR‐Dextran, using Immunofect MG beads, indicates that transient holes of 5.4 nm in diameter or larger are formed during immunoporation. The efficiencies of transfection of lymphocytes transfected with vectors coding for EGFP and lacZ were found to be within the range of 15–30% with high levels of cell viability of more than 90%. In addition, it was observed that mononuclear cells stimulated with PHA expressed transfected reporter genes with a higher efficiency. In conclusion, these results demonstrate that immunoporation using Immunofect MG beads can be used for the efficient transfection of primary lymphocytes with DNA or other macromolecules.

The proteasome is the major cellular proteolytic machinery responsible for the degradation of both normal and damaged proteins. Proteasomes play a fundamental role in retaining cellular homeostasis. Alterations of proteasome function have been recorded in various biological phenomena including aging. We have recently shown that the decrease in proteasome activity in senescent human fibroblasts relates to the down-regulation of beta-type subunits. In this study we have followed our preliminary observation by developing and further characterizing a number of different human cell lines overexpressing the beta5 subunit. Stable overexpression of the beta5 subunit in WI38/T and HL60 cells resulted in elevated levels of other beta-type subunits and increased levels of all three proteasome activities. Immunoprecipitation experiments have shown increased levels of assembled proteasomes in stable clones. Analysis by gel filtration has revealed that the recorded higher level of proteasome assembly is directly linked to the efficient integration of "free" (not integrated) alpha-type subunits identified to accumulate in vector-transfected cells. In support we have also found low proteasome maturation protein levels in beta5 transfectants, thus revealing an increased rate/level of proteasome assembly in these cells as opposed to vector-transfected cells. Functional studies have shown that beta5-overexpressing cell lines confer enhanced survival following treatment with various oxidants. Moreover, we demonstrate that this increased rate of survival is due to higher degradation rates following oxidative stress. Finally, because oxidation is considered to be a major factor that contributes to aging and senescence, we have overexpressed the beta5 subunit in primary IMR90 human fibroblasts and observed a delay of senescence by 4-5 population doublings. In summary, these data demonstrate the phenotypic effects following genetic up-regulation of the proteasome and provide insights toward a better understanding of proteasome regulation.

Immunoporation is a novel method of cell transfection based upon the use of a new type of beads, Immunofect beads, that can be targeted to make holes in different types of cells depending on the type of bead used. It is known that the efficiency of transfection of cells by some techniques can be affected by the presence of serum and another important factor that appears to affect transfection efficiency and cell viability is the osmolarity of the transfection medium. This report presents studies on the effects of serum and varying osmolarity on the efficiency of transfection using immunoporation. The results clearly indicate that in hypertonic media the presence of serum decreases the efficiency of transfection. In the case of osmolarity, increasing the osmolarity of the immunoporation medium increases the efficiency of transfection but above about 650 mOsm this increasing efficiency is offset by the much lower viability of the cells.

Cell transfection is now a central technique in molecular biology and an essential prerequisite for gene therapy. Here we describe how beads coated with antibodies and bound to specific cell-surface transmembrane proteins can create holes in cells when the beads are removed, allowing transfection of the cells with DNA or other macromolecules. This unique targeted transfection of cells by immunoporation is very efficient and results in minimal cell death.

Abstract 3183 Background: The introduction of iron chelation treatment has led to a significant improvement in morbidity and overall survival in patients with transfusion-dependent anemias. Deferasirox is a once-daily, oral iron chelator approved for the treatment of transfusional iron overload in both adult and pediatric patients. The efficacy and safety of deferasirox in a variety of transfusion-dependent anemias has been established in numerous Phase II/III clinical trials. Since most patients with transfusion-dependent anemias require lifelong iron chelation therapy, there is a need to assess the long-term safety of deferasirox in both adult and pediatric patients. Aim: To assess the safety profile of deferasirox in patients with transfusional iron overload in a real-world clinical setting. To further investigate the safety profile of deferasirox in patients with congenital erythrocyte disorders and transfusional iron overload, with ferritin levels Methods: Between July 2009 and September 2010, 85 patients with transfusion-induced iron overload treated with deferasirox as per the approved product labeling were enrolled in the study. These data represent the 24-week planned interim analysis of a 12-month observational study on deferasirox safety profile in the treatment of pediatric and adult patients with transfusion-dependent anemias who were newly-treated with deferasirox. Safety was evaluated through the monitoring and recording of all adverse events and serious adverse events, as well as routine laboratory testing, including hematology, blood chemistry and hepatic function assessments. Results: The population had a median age of 37.6 years (range: 5.3–61.4) and a female to male ratio of 1.3. Beta-thalassemia (67.1%) was the most common transfusion-dependent anemia, followed by thalassemia intermedia requiring periodic transfusions (20.0%) and sickle cell anemia (12.9%). Mean baseline ferritin levels were 1502.1±870.5 (pediatric group: 1480.2±522.8 and adult group: 1503.6±891.4), while 53 out of the 85 patients (62.4%) had serum ferritin level above 1000 ng/ml. Mean baseline liver T2* value was 10.4±9.7 ms; 44.4% of patients demonstrated minimal liver iron deposition (MRI T2* > 6.3 ms), 51.4% had mild to moderate liver iron overload (T2* ≤ 6.3 ms), and 4.2% had severe liver iron overload (T2* 54 (63.5%) of patients analysed had been pre-treated with iron chelators and 31 (36.5%) were chelation-naïve. The initial average daily dose of deferasirox was 25.9±4.8 mg/kg, and 70.6% of patients had no dose modification during the 24-week follow-up period. A statistical significant decrease in median serum ferritin levels was observed by Week 24 (mean absolute change from baseline:-214.5 ng/mL; p=0.009) [Figure 1]. No statistically significant changes were observed in creatitine levels, creatinine clearance and transaminases by Week 24 [Figure 1]. 37 ADRs were reported by 17 patients (20%) over the 24-week period. Among the most frequently observed ADRs (>5%) were epigastralgia reported by 7.1% of patients (6/85) and loose stools/diarrhoea by 5.9% of patients (5/85). The majority of ADRs reported (nevents=25; 67.6%) were graded as mild in severity, while 21.6% (nevents=8) were graded as moderate and 10.8% (nevents=4) as severe. Most ADRs (nevents=31; 83.8%) resulted in full recovery by Week 24. The overall incidence of SADRs was as low as 1.2% (in particular one patient experienced severe epigastralgia and upper extremity pain which resulted in her withdrawal from the study after four months of treatment). The all-cause discontinuation rate was 9.4% (8/85), while only two patients (2.4%) discontinued the study therapy due to ADR; 1 patient due to increased transaminase levels and 1 patient due to the aforementioned SADR. Conclusions: These data highlight the safety profile of deferasirox in both adult and pediatric patients; the regular monitoring of serum ferritin levels as well as other iron-overload parameters and transfusion requirements play a major role in determining and optimizing the outcome of iron chelation therapy. Disclosures: Ladis: Novartis Hellas S.A.C.I.: Investigator participating in a trial sponsored by Novartis. Drossou:Novartis Pharmaceuticals: Investigator participating in a trial sponsored by Novartis. Vini:Novartis Pharmaceuticals: Investigator participating in a trial sponsored by Novartis. Athanasiou-Metaxa:Novartis Hellas S.A.C.I.: Research Funding. Oikonomou:Novartis Hellas S.A.C.I.: Investigator participating in a trial sponsored by Novartis. Vlachaki:Novartis Hellas S.A.C.I.: Investigator participating in a trial sponsored by Novartis. Tigka:Novartis Hellas S.A.C.I.: Employment. Tzavelas:Novartis Hellas S.A.C.I.: Employment. Liakopoulou:Novartis Hellas S.A.C.I.: Investigator participating in a trial sponsored by Novartis. Adamopoulos:Novartis Hellas S.A.C.I.: Investigator participating in a trial sponsored by Novartis. Kattamis:Novartis Hellas S.A.C.I.: Honoraria, Membership on an entity's Board of Directors or advisory committees.