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Physiologic loss of telomerase activity in adult life determines progressive telomere length (TL) shortening. Inflammation and oxidative damage are established causes of TL loss; moreover, males have shorter telomeres compared with females. Despite these notions, mechanisms regulating TL maintenance are poorly defined. Because umbilical cord blood (UCB) cells harbor very long telomeres, not yet exposed to environmental damages, UCB transplantation (UCBT) provides a unique experimental setting to study determinants of TL in humans. TL dynamics were analyzed on peripheral blood mononuclear cells (MNCs) from 36 patients (median age, 42 years) undergoing UCBT. TL was studied at a median of 20 months after UCBT. A significantly longer TL (mean, 8698 bp; range, 6521 to 11,960) was documented in UCBT recipients compared with age-matched healthy control subjects (mean, 7396 bp; range, 4375 to 11,108; P.01). Among variables potentially influencing TL maintenance, including recipient features, graft type, transplant procedure, and engraftment kinetics, only donor-recipient gender combination was associated with TL, with the longest TL in women receiving male UCB (mean, 10,063 bp; range, 8381 to 11,960). To further investigate this trend, telomerase activation was assessed in vitro. Experiments showed that telomerase subunits were preferentially upregulated in male-derived bone marrow MNCs exposed ex vivo to estradiol as compared with female MNCs. This implies an increased sensitivity of male-derived MNCs to telomerase activation induced by estradiol. The results suggest that extrinsic and modifiable factors such as hormonal status and female milieu could be major determinants of TL in humans, providing the rationale for investigating hormonal-based approaches to counteract telomere erosion and aging-related diseases.

CD40/CD40 ligand (CD40L) dyad, a co-stimulatory bi-molecular complex involved in the adaptive immune response, has also potent pro-inflammatory actions in haematopoietic and non-haematopoietic cells. We describe here a novel role for soluble CD40L (sCD40L) as modifier of glomerular permselectivity directly acting on glomerular epithelial cells (GECs). We found that stimulation of CD40, constitutively expressed on GEC cell membrane, by the sCD40L rapidly induced redistribution and loss of nephrin in GECs, and increased albumin permeability in isolated rat glomeruli. Pre-treatment with inhibitors of CD40-CD40L interaction completely prevented these effects. Furthermore, in vivo injection of sCD40L induced a significant reduction of nephrin and podocin expression in mouse glomeruli, although no significant increase of urine protein/creatinine ratio was observed after in vivo injection. The same effects were induced by plasma factors partially purified from post-transplant plasma exchange eluates of patients with focal segmental glomerulosclerosis (FSGS), and were blocked by CD40-CD40L inhibitors. Moreover, 17 and 34 kDa sCD40L isoforms were detected in the same plasmapheresis eluates by Western blotting. Finally, the levels of sCD40Lwere significantly increased in serum of children both with steroid-sensitive and steroid-resistant nephrotic syndrome (NS), and in adult patients with biopsy-proven FSGS, compared to healthy subjects, but neither in children with congenital NS nor in patients with membranous nephropathy. Our results demonstrate that sCD40L directly modifies nephrin and podocin distribution in GECs. Moreover, they suggest that sCD40L contained in plasmapheresis eluates from FSGS patients with post-transplant recurrence may contribute, presumably cooperating with other mediators, to FSGS pathogenesis by modulating glomerular permeability.

Various agents, including chemotherapeutic drugs, can induce cell senescence. However, the mechanisms involved in the aging pathway, particularly the stress that chemotherapy imposes on telomeres, are still undefined. To address these issues, human mesenchymal stem cells (MSCs) were assessed as target cells to investigate the initiation of the aging process by chemotherapy. The MSCs were obtained from bone marrow (BM) cells from normal adults and grown in the presence of platelet lysates. Cultured MSCs were identified for immunophenotype, and for growth and differentiation properties. The MSCs were exposed to 10 nM doxorubicin and 500 ng/mL etoposide, sublethal doses that induce DNA double-stranded breaks. Telomere length (TL) was assessed by flow-fluorescence in situ hybridization and Southern blotting. Initial TL shortening was detectable in MSCs at 5 days after drug exposure, with progressive reduction compared with untreated cells at 7, 14, 21, and 28 days in culture. After a single exposure, MSCs were unable to regain the lost telomere sequences for up to 28 days in culture. The ATM phosphorylation was documented early after drug exposure, while no telomerase activation was observed. Chemotherapy-induced TL shortening was associated with reduced clonogenic activity in vitro and accelerated adipose differentiation. Analogous behavior in the differentiation pattern was observed in naturally aged MSCs. These results indicate that cultured MSCs represent a useful cellular model to investigate novel drugs that may favor or, conversely, might prevent TL loss in human stem cells. The TL shortening is a permanent signature of previous chemotherapy-mediated DNA damage, and predicts impaired proliferative and differentiation potential.

Objectives : Thrombopoietin (TPO) is a humoral growth factor that primes platelet activation in response to several agonists. We recently showed that TPO enhances platelet activation in unstable angina and sepsis. Aim of this study was to investigate the role of TPO in platelet function abnormalities described in cigarette smokers. Methods : In a case–control study we enrolled 20 healthy cigarette smokers and 20 nonsmokers, and measured TPO and C-reactive protein (CRP), as well as platelet–leukocyte binding and P-selectin expression. In vitro we evaluated the priming activity of smoker or control plasma on platelet activation, and the role of TPO in this effect. We then studied the effects of acute smoking and smoking cessation on TPO levels and platelet activation indices. Results : Chronic cigarette smokers had higher circulating TPO levels than nonsmoking controls, as well as increased platelet–leukocyte binding, P-selectin expression, and CRP levels. Serum cotinine concentrations correlated with TPO concentrations, platelet–monocyte aggregates and P-selectin expression. In addition, TPO levels significantly correlated with ex vivo platelet–monocyte aggregation and P-selectin expression. In vitro , the plasma from cigarette smokers, but not from nonsmoking controls, primed platelet–monocyte binding, which was reduced when an inhibitor of TPO was used. We also found that acute smoking slightly increased TPO levels, but did not affect platelet–leukocyte binding, whereas smoking cessation induced a significant decrease in both circulating TPO and platelet–leukocyte aggregation. Conclusion : Elevated TPO contributes to enhance platelet activation and platelet–monocyte cross-talk in cigarette smokers.

The injury of podocytes is associated with alterations of the glomerular size-selective barrier to proteins. In this study, oxidized LDL (oxLDL) but not native LDL induced apoptosis in human cultured podocytes and reduced Akt activity and P-Akt/Akt ratio. Moreover, oxLDL-induced redistribution and loss of nephrin, an adhesion molecule specific for the glomerular slit diaphragm. Nephrin reduction was preceded by inhibition of nephrin tyrosine phosphorylation and of its association with p85 phosphatidylinositol 3-kinase (PI3K). Moreover, three different statins, mevastatin, pravastatin, and simvastatin, inhibited in a dose-dependent manner apoptosis and loss of nephrin induced by oxLDL by stimulating Akt activity. In addition, simvastatin significantly increased the expression of nephrin protein and mRNA by podocytes. The protective effects of statins were blocked by treatment of podocytes with two unrelated pharmacologic inhibitors of PI3K, LY294002 and wortmannin, suggesting a role for PI3K, and by mevalonate, indicating dependency on HMG-CoA reductase activity. Statins directly stimulated Akt phosphorylation ad activity. Finally, oxLDL induced a retraction of cultured podocytes and an increase in the albumin diffusion across their monolayer that was inhibited by treatment with statins. In conclusion, statins reduced the oxLDL-induced apoptosis and loss of nephrin in glomerular podocytes. The statin-induced Akt activation may protect from the loss of nephrin by an inhibition of its redistribution and shedding and by a stimulation of its synthesis. These data provide a rationale for the anti-proteinuric effect of statins.

BACKGROUND AND PURPOSE:Thrombopoietin (TPO), a growth factor primarily involved in thrombopoiesis may also have a role in the pathophysiology of sepsis. In patients with sepsis, indeed, TPO levels are markedly increased, with disease severity being the major independent determinant of TPO concentrations. Moreover, TPO increases and correlates with ex vivo indices of platelet activation in patients with burn injury upon sepsis development, and may contribute to depress cardiac contractility in septic shock. Still, the role of TPO in sepsis pathophysiology remains controversial, given the protective role of TPO in other experimental disease models, for instance in doxorubicin-induced cardiotoxicity and myocardial ischemia/reperfusion injury. The aim of our study was to define the contribution of TPO in the development of organ damage induced by endotoxemia or sepsis, and to investigate the effects of inhibiting TPO in these conditions. METHODS:We synthesized a chimeric protein able to inhibit TPO, mTPOR-MBP, and studied its effect in two murine experimental models, acute endotoxemia and cecal ligation and puncture (CLP) model. RESULTS:In both models, TPO levels markedly increased, from 289.80±27.87 pg/mL to 465.60±45.92 pg/mL at 3 hours in the LPS model (P

The purpose of this study was to investigate telomere length (TL) in Ph-negative chronic myeloproliferative neoplasms (Ph-neg-CMNs), and the possible association of TL with disease progression and hydroxycarbamide (HU) treatment. TL was analyzed in peripheral blood samples from 239 patients with Ph-neg-CMNs, including polycythemia vera (PV), essential thrombocythemia and myelofibrosis (MF), and compared with age-matched healthy control subjects (CTR), along with some cases of secondary erythrocytosis (SE). More than half of the patients with CMN received at least 1 year of cytoreduction, mainly HU, before TL analysis. JAK2 mutation analysis was performed as well. TL was significantly shortened in patients with CMN compared with CTR (p0.0001). PV and MF showed the most pronounced decrease (p0.0001), whereas both essential thrombocythemia and SE showed no significant difference in TL compared with CTR. A short TL correlated with JAK2-V617F allele burden greater than 50% (p = 0.0025), age (p = 0.0132) and diagnosis of PV (p = 0.0122). No correlation was found with disease duration, history of thrombosis, cytoreductive treatment, antiaggregation agents, adverse cytogenetics, phlebotomies, or time to evolution to MF. In summary, TL is distinctly shortened in PV and MF, and it inversely correlates with JAK2V617F allele burden. In addition, HU is unlikely to contribute to telomere erosion. Lastly, PV and SE significantly differ in TL. Therefore, TL could be an additional diagnostic marker to identify and monitor Ph-neg-CMN patients.

Thrombopoietin (TPO) is a humoral growth factor that has been shown to increase platelet activation in response to several agonists. Patients with sepsis have increased circulating TPO levels, which may enhance platelet activation, potentially participating to the pathogenesis of multi-organ failure. Aim of this study was to investigate whether TPO affects myocardial contractility and participates to depress cardiac function during sepsis. We showed the expression of the TPO receptor c-Mpl on myocardial cells and tissue by RT-PCR, immunofluorescence and western blotting. We then evaluated the effect of TPO on the contractile function of rat papillary muscle and isolated heart. TPO did not change myocardial contractility in basal conditions, but, when followed by epinephrine (EPI) stimulation, it blunted the enhancement of contractile force induced by EPI both in papillary muscle and isolated heart. An inhibitor of TPO prevented TPO effect on cardiac inotropy. Treatment of papillary muscle with pharmacological inhibitors of phosphatidylinositol 3-kinase, NO synthase, and guanilyl cyclase abolished TPO effect, indicating NO as the final mediator. We finally studied the role of TPO in the negative inotropic effect exerted by human septic shock (HSS) serum and TPO cooperation with TNF-alpha and IL-1beta. Pre-treatment with the TPO inhibitor prevented the decrease in contractile force induced by HSS serum. Moreover, TPO significantly amplified the negative inotropic effect induced by TNF-alpha and IL-1beta in papillary muscle. In conclusion, TPO negatively modulates cardiac inotropy in vitro and contributes to the myocardial depressing activity of septic shock serum.

Summary. Background: Thrombopoietin (TPO) is a humoral growth factor that does not induce platelet aggregation per se, but enhances platelet activation in response to several agonists. Circulating levels of TPO are increased in patients with sepsis and are mainly related to sepsis severity. Objectives: To investigate the potential contribution of elevated TPO levels in platelet activation during burn injury complicated or not by sepsis. Methods: We studied 22 burned patients, 10 without and 12 with sepsis, and 10 healthy subjects. We measured plasma levels of TPO, as well as leukocyte-platelet binding and P-selectin expression. The priming activity of plasma from burned patients or healthy subjects on platelet aggregation and leukocyte-platelet binding, and the role of TPO in these effects were also studied in vitro. Results: Burned patients without and with sepsis showed higher circulating TPO levels and increased monocyte-platelet binding compared with healthy subjects. Moreover, TPO levels, monocyte-platelet binding and P-selectin expression were significantly higher in burned patients with sepsis than in burned patients without sepsis. In vitro, plasma from burned patients without and with sepsis, but not from healthy subjects, primed platelet aggregation, monocyte-platelet binding and platelet P-selectin expression. The effect of plasma from burned patients with sepsis was significantly higher than that of plasma from burned patients without sepsis. An inhibitor of TPO prevented the priming effect of plasma from burned patients. Conclusions: Increased TPO levels may enhance platelet activation during burn injury and sepsis, potentially participating in the pathogenesis of multi-organ failure in these diseases.

Objective To determine whether HIV-1 Tat may directly alter glomerular permeability in HIV-associated nephropathy (HIVAN). Design Heavy proteinuria is a hallmark of HIVAN. The slit diaphragm is the ultimate glomerular filtration barrier critical for maintaining the efficiency of the ultrafiltration unit of the kidney. In this study, we evaluated the direct effect of Tat protein on the permeability of isolated glomeruli and on the expression of nephrin, the main slit diaphragm component, by human cultured podocytes. Methods Permeability was studied by measuring the permeability to albumin in isolated rat glomeruli. We also evaluated the expression of nephrin in human cultured podocytes by using immunofluorescence and Western blot. Results We found that Tat increased albumin permeability in isolated glomeruli, and rapidly induced the redistribution and loss of nephrin in cultured podocytes. Pretreatment of glomeruli and podocytes with blocking antibodies showed that Tat reduced nephrin expression by engaging vascular endothelial growth factor receptors types 2 and 3 and the integrin alphavbeta3. Pre-incubation of podocytes with two platelet-activating factor (PAF) receptor antagonists prevented the loss and redistribution of nephrin induced by Tat, suggesting that PAF is an intracellular mediator of Tat action. Tat induced a rapid PAF synthesis by podocytes. When podocytes transfected to overexpress PAF-acetylhydrolase, the main catabolic enzyme of PAF, were stimulated with Tat, the redistribution and loss of nephrin was abrogated. Conclusion The present results define a mechanism by which Tat may reduce nephrin expression in podocytes, thus increasing glomerular permeability. This provides new insights in the understanding of HIVAN pathogenesis.

The presence of circulating plasma factors (PF) altering renal permeability to proteins has been previously described in patients with focal segmental glomerulosclerosis (FSGS). Since these patients show reduced nephrin and podocin expression at renal biopsy, we evaluated the effect of serum and PF from patients with FSGS on nephrin and podocin expression in human podocytes. We studied 7 sera from patients with steroid-resistant FSGS, 3 from patients with nephrotic syndrome caused by non-immune disease, and 6 from healthy subjects. PF was prepared from plasmapheresis eluates of 2 patients with post-transplant recurrence of FSGS. Purification procedure was based on protein A Sepharose chromatography and differential precipitation in ammonium sulphate. Nephrin and podocin expression was semi-quantitatively evaluated by immunofluorescence. We found that serum and PF from FSGS patients rapidly induced redistribution and loss of nephrin in podocytes. This effect was associated with cytoskeleton redistribution and inhibited by cytochalasin B and sodium azide. On the contrary, podocin expression was unchanged after incubation with serum and PF from FSGS patients for short periods, but markedly reduced at 24 h. Our results demonstrate that serum and PF from FSGS patients may directly affect nephrin and podocin in human podocytes, thus providing new insights into the mechanisms causing proteinuria in FSGS.

Introduction TNF-related apoptosis-inducing ligand (TRAIL) is a protein functioning as a ligand that induces the process of cell death. TRAIL has been shown to kill in vitro a wide variety of tumor cells with minimal effects on normal cells. Despite its in vitro activity, recombinant soluble TRAIL has so far shown limited efficacy in vivo. In contrast, recent reports have shown that significant apoptosis can be observed both in vitro and in vivo when TRAIL is expressed on the cell membrane (mTRAIL). A further innovation might be the delivery of bioactive proapoptotic TRAIL through its expression by extracellular vescicles (EVs), the nanovesicular organelles secreted by cells. In fact, EVs are viewed as an effective tool for intercellular cross-talk and receptor discharge. The trans-membrane expression of TRAIL ligand within the double layer exosomal membrane may induce a more potent death signal when compared with the soluble molecule. Material and Methods Mesenchymal Stromal Cells (MSC) from bone marrow were cultured in vitro and used for EVs production. Cultured MSC in 75 cm2 flasks, at 80% confluence were infected with a lentivector encoding TRAIL, maintained in culture, and cell-supernatants repeatedly collected over several days, ultracentrifugated, with EVs-containing pellet harvested in PBS. EVs were produced also from uninfected MSC as control (EVs-CTRL). EVs were characterized by flow cytometry for expression of MSC markers and mTRAIL, EV size was evaluated by NanoSight technology. Total protein concentration was used to quantify EVs, Western Blot analysis was performed to characterize membrane-bound TRAIL. In vitro analysis was performed on SU-DHL-4 (human B cell lymphoma) and MEL-1300 (human melanoma) cell lines, exposed for 24 hours to 20-100 μg/ml EVs-TRAIL or EVs-CTRL. Annexin/propidium iodide assay was used to quantify apoptotic/necrotic cells. For the in vivo assessments, SU-DHL-4 and MEL-1300 cells were transduced with Luc-Lentiviral particles to obtain Luciferase positive cell lines. These cells were used to engraft NOD scid gamma (NSG) mice (2x106 SU-DHL-4 and 3x105 MEL-1300 cells for each subcutaneous injection point). To visualize tumor cells, mice were injected intraperitoneum with luciferin and analyzed with the Xenogen system. Mice bearing subcutaneous tumor nodules received single intravenous injections of 100, 200, 300 µg or multiple (x 3) 200 µg injections of either EVs-TRAIL or EVs-CTRL. Results FACS analysis showed strong TRAIL expression on EVs from TRAIL-infected MSC compared to EVs-CTRL, with a high proportion of positive particles (median 85%, range 78-93). In addition, EVs-TRAIL displayed MSC membrane markers, i.e. CD 105, CD 90, CD73 and CXCR4. Western Blot analysis under non-reducing conditions showed the presence of TRAIL ligand, with strong prevalence of dimeric TRAIL isoform (barely detectable the trimeric isoform, undetectable monomeric isoforms). NanoSight analysis revealed that EVs had a variable size, up to approximately 400 nm in diameter, with a predominant peak at 273 nm. A strong and dose-dependent cytotoxic effect was observed on SU-DHL-4 cells exposed to EVs-TRAIL (annexin/PI+ve cells: up to 87% for 100 μg/ml EVs-TRAIL), compared to EVs-CTRL exposure (15% Annexin/PI+ve cells for 100 μg/ml EVs-TRAIL). A similar, albeit less pronounced in vitro cytotoxic effect of EVs-TRAIL was observed on the melanoma MEL-1300 cell line. The anti-tumor effect was remarkably strong when EVs-TRAIL were injected in vivo in mice bearing either SU-DHL-4 or MEL-1300 nodules. A marked reduction of the tumor luminescence from 1.2x1010 photon/sec to Conclusion EVs isolated from genetically engineered TRAIL-expressing MSC: i. do express mTRAIL; ii. display potent antitumor activity, inducing extensive apoptosis/necrosis both in vitro and in vivo in animal models bearing lymphoma and melanoma nodules. Thus, EVs-TRAIL may represent a promising strategy for delivering pro-apoptotic signals to tumor cells. Moreover, the Results could pave the way to the use of EVs for therapeutic purposes. Disclosures: No relevant conflicts of interest to declare.

Abstract 4094 Background: Telomeres are linear DNA structures at the ends of chromosomes that help to maintain genome stability. In the absence of effective telomere-protecting mechanisms, chromosomes lose a few base pairs (bp) of their telomeric DNA at each mitotic division and thus telomere length (TL) is also considered a marker of cell aging. In the hematopoietic system, TL of both early and committed progenitors progressively shortens with age, as in other highly replicating tissues. Moreover, many studies demonstrated that high proliferative stresses, including bone marrow (BM) regeneration after transplantation, cause a non-physiological TL shortening. Indeed, recent studies suggest that post-autograft TL correlates with TL of grafted material (Ruella et al, 2010). In order to address this issue, TL has been evaluated in a series of adult subjects engrafted with umbilical cord blood (UCB), a graft source characterized by cells harboring very long telomeres. Aim of the study was to verify whether despite the high replicative stress during engraftment, UCB hematopoietic stem cells may renew the recipient BM, maintaining the biologic characteristics of the graft. Patients and Methods: TL was evaluated on peripheral blood (PB) cells from 20 adult recipients of umbilical cord blood transplant (UCBT). They had been transplanted due to acute leukemia (n=12), multiple myeloma (n=2), non-Hodgkin's Lymphoma (n=3) or chronic myeloproliferative disease (=3). TL analysis was performed in absence of signs of the previous disease and at complete hematological recovery, at a median of 9 mos. (range 1–73) since UCBT. At TL analysis, cell blood counts revealed median values of 7,540 white blood cells/μL (range 2,870-18,800), 11.9 gr% of hemoglobin (range 8.3–14), and 144,000 platelets/μL (range 41,000–351,000). TL was evaluated on both whole PB cells and separated cell populations, i.e. MNC and granulocytes, obtained following Ficoll gradient cell separation. TL was also determined on a series of 47 UCB samples, including samples from cord blood units employed as graft material for four patients of the present series. TL was determined on DNA extracts by Southern blot analysis, according to well established procedures. Briefly, DNA fragments were separated by agarose gel electrophoresis. The Telomere Length Assay Kit (Roche Diagnostic) was used for the hybridization phase and for each telomere smear, mean terminal restriction fragment (TRF) lengths and the point of maximum signal intensity defining the highest concentration of telomere repeats were calculated using Quantity One software (Bio-Rad Laboratories, UK). Results: Overall, TL in PB cells following UCBT showed a rapid fall compared to TL values that characterize cord blood cells. In our series of 47 UCB samples, the median TL-TRF on MNC is 9,171 bp (range 6,573-13,695), whereas MNC from our UCBT recipients showed a median TL-TRF of 8,689 bp (range 5,982-11,149) (p=0,055). When values were distributed according to the age of UCBT recipients, TL remained higher than TL obtained from a large series of hundreds of normal control subjects. Indeed, median TL-TRF on MNC from healthy controls is 6,951 bp (range 4,375-10,467), significantly shorter compared to MNC from UCBT recipients, as shown in Figure 1. Similar results were obtained when TL was assessed on whole PB cells and on separated granulocytes. In fact, the most marked difference in TL between UCBT recipients and normal controls was observed on granulocytes. This was particularly clear among subjects aged >40 yrs. As shown in Figure 2, median TL-TRF on granulocytes from UCBT recipients aged 40–65 yrs. was 9,138 bp (range 7,644 – 11,138) compared to 6,857 bp (range 5,773 – 10,946) of granulocytes from age-matched normal controls. Overall, PB cells from subjects grafted with UCB showed a TL slightly longer than in the youngest control population of 0–20 yrs. of age. Lastly, no difference in TL was observed according to time elapsed since transplant, with analogous TL values in subjects evaluated at less or more than 6 months since UCBT. Conclusions: The results indicate that despite the high replicative stress during BM engraftment, UCBT regenerate a hematopoietic system characterized by long TL. This suggests that in the transplant setting the use of young donor cells such as UCB may replace patient BM with not only a healthy but also a biologically young hematopoietic system. Disclosures: Rambaldi: Hoffman-La Roche: Consultancy, Honoraria.

Abstract 1620 Introduction Patients with lymphoproliferative disease very often require treatments with chemotherapy. Several recent reports suggest that the exposure of hematopoietic cells to DNA-damaging substances, such as chemotherapeutic drugs, may trigger the ageing process and induce premature cell ageing. A good indicator of cell replication history and thus of cell ageing is the length of telomeres. Indeed, telomere shortening and/or telomere dysfunction have been documented in patients receiving chemotherapy. The loss of telomere sequences has been linked to the increased risk of developing secondary malignancy in subjects previously treated with chemotherapy. However, it is still unexplained whether telomere loss is secondary to direct damage of bone marrow (BM) cells by cytotoxic drugs, or merely the consequence of increased BM cell proliferation after exposure to chemotherapy. Moreover, the time course for the onset of chemotherapy-induced telomere shortening and the cell types mainly involved in the premature cell aging induced by chemotherapy remain to be elucidated. In the present study changes in telomere length (TL) following chemotherapy were evaluated both in vivo and in vitro. Main aims of the study were: i. to define whether TL shortening following chemotherapy is reversible or permanent; ii. to verify whether TL shortening is a phenomenon induced by extensive chemotherapy treatments or it may occur even after minimal drug exposures; iii. to identify possible cell populations that are particularly susceptible to drug-induced telomere loss. Patients and methods Mononuclear Cells (MNC) or granulocytes were obtained from BM and/or Peripheral blood (PB) cells from 31 lymphoma patients treated with high-dose chemotherapy and autograft and from 14 lymphoma patients undergoing chemotherapy (6 CHOP, 1 CVP, 1 MINE, 1 DHAP and 5 ABVD). Overall, 27 patients of the autograft and 13 patients of the conventional chemotherapy groups were at their first treatment line. Median age of patients was 45 years. TL was assessed on granulocytes and MNC by Southern Blotting, as previously reported (Ricca et al, Leukemia 2005). In vitro studies were performed on cultured mesenchymal stem cells (MSCs). Briefly, MNC were obtained from BM of normal volunteers or patients undergoing routine diagnostic procedures; cells were seeded in MEM-alpha medium and 10% platelet lysates and fed at 3–4 day intervals; cultured MSC were identified for positivity of CD105, CD90, CD29, CD44 and then assayed for response to DNA-damaging drugs between the second and the third passage in culture. Two chemotherapeutic drugs were employed, Doxorubicin (Doxo) and etoposide (Eto). Cells were incubated for 2 hours with decreasing doses of the tested drugs, and 10 nM Doxo and 500 ng/ml Eto were the highest doses of the drugs that were used without any distress on cell proliferation and cell viability. The 2-hour exposure to chemotherapy was repeated at 7 day intervals up to four times. TL was evaluated both by flow-fish and southern-blot analysis. Results A marked reduction in TL was detected in all patients undergoing autograft in PB granulocytes and in BM cells, compared to age-matched controls; the degree of TL loss remained detectable even in patients up to 10 yrs. since autograft. For patients receiving conventional chemotherapy, a significant TL shortening could be detected in granulocyte obtained after chemotherapy compared to pre-treatment values (p=0.029), while no significant variations could be documented in MNC. TL shortening was detectable already after the first chemotherapy course in six patients and at the second in four patients. A marked TL shortening occurred in cultured MSC after exposure to sub-lethal doses of Doxo and Eto. Initial TL shortening was detectable already at 5 days after drug exposure, with progressive reduction compared with untreated cells at 7, 14, 21, and 28 days in culture. Following a single exposure, MSCs were unable to regain the lost telomere sequences for up to 28 days in culture. Conclusions The results indicate that TL shortening: i. is a permanent signature of the previous DNA damage in BM cells exposed to chemotherapy; ii. is a phenomenon that can be detected early following chemotherapy exposure, even with low drug dosages; iii. can be most easily detectable in myeloid cells, in particular in granulocytes, although also BM mesenchymal cells may be susceptible to drug-induced telomere loss. Disclosures: No relevant conflicts of interest to declare.

Abstract 4776 Introduction. A good indicator of the rate of cell ageing is the length of telomeres, the long tandem repeat sequences at the end of chromosomes. Telomere length (TL) in humans decreases at each somatic cell division, and undergoes progressive shortening with increasing age. Indeed, cell proliferation has been considered the primary factor responsible of TL shortening and ultimately of the cell ageing process. Exposure to DNA-damaging substances has been proposed as an alternative and relevant factor involved in the cell ageing process. In spite of the number of studies in both human and animal models, few information is available on the role of both cell proliferation and DNA damage in normal human stem cells. The present study addressed this issue by investigating the ageing process in cultured Bone-Marrow (BM) human Mesenchymal Stem Cells (MSCs). Aim of the study has been to investigate whether and how exposure to toxic agents may affect the rate of TL shortening in cultured MSCs. Methods. 1. MSCs were cultured from human mononuclear BM cells, obtained from normal volunteers or patients undergoing routine diagnostic procedures; cells were seeded in MEM-alpha medium containing heparin and 10% platelet lysates and fed at 3–4 day intervals, until they reached confluence; they were then detached with trypsin-EDTA solution and reseeded for the experimental tests. Cultured MSC were identified for positivity of CD105, CD90, CD29, CD44 and negativity for CD14, CD34 and CD45 by flow cytometry; 2. cultured MSC were assayed for response to DNA-damaging drugs between the second and the third passage in culture. Two chemotherapeutic drugs were employed to induce DNA damage: Doxorubicin (Doxo) and etoposide (Eto). Cells were incubated for 2 hours with decreasing doses of the tested drugs, and 10 nM Doxo and 500 ng/ml Eto were the highest doses of the drugs that were used without any distress on cell proliferation and cell viability. The 2-hour exposure to chemotherapy was repeated at 7 day intervals up to four times; 3. telomere was evaluate in MSCs before (day 0 or T0) and after exposure to chemotherapeutic drugs, i.e. at 7 – 14 –21 and 28 days of culture since drug exposure. TL was determined by the cytofluorimetric Flow-fish assay and by southern-blot analysis; 4. MSC were also cultured in differentiating medium in order to induce osteogenic and adipogenic differentiation, evaluated by Alizarin red and Oil red staining, respectively; 5. lastly, both ATM phosphorilation and telomerase activation were assessed, by Western blot analysis and the TRAP assay, respectively. Results. 1. as expected, TL progressively decreased after 7 days in culture in untreated (NT) MSC (88,8% compared to T0); TL was further shortened as long as the cells were maintained in culture; 2. a marked TL shortening occurred in MSC already at day 7 after exposure to sub-lethal doses of Doxo and Eto, with a mean of 81,7% and 79,6% compared to T0, respectively. Indeed, a minimum of 5 days in culture was required to identify the earliest signs of telomere loss. Again, further TL shortening was observed later on and the difference in TL between NT and chemotherapy-exposed cells progressively increased: compared to T0, TL resulted of 82 % and 74% in NT cells, whereas TL was 68% and 54% in Doxo-treated, and 70% and 62% in Eto-treated, at week 3 and 4, respectively; 3. a single 2-hr exposure to Doxo or Eto induced a TL loss compared to NT cells, that was maintained unchanged at long-term in culture; 4. when MSC were assayed in differentiation inducing medium, cells exposed to chemotherapy displayed enhanced adipogenic and osteogenic differentiation compared to NT cells; 5. ATM was strongly phosphorilated following exposure to Doxo and Eto compared to NT MSC; ATM phosphorilation preceded TL loss; by contrast, telomerase was unaffected by chemotherapy exposure. Conclusions. i. human cultured MSCs exposed to chemotherapeutic drugs offer a simple and reliable means to investigate the correlation between DNA damage and cell ageing; ii. telomere loss is clearly detectable in MSCs already after 7 days following exposure to Doxo and Eto and remains detectable at long-term in culture as a permanent signature of the previous DNA damage; iii. the in vitro observation suggests that not only hematopoietic cells but also stromal cells, including MSCs, may experience relevant DNA damages following in vivo treatments with chemotherapeutic drugs. Disclosures: No relevant conflicts of interest to declare.