Aim: To evaluate the reliability of linear measurements in virtual models by comparing measurements performed on virtual models obtained from alginate impression scans, plaster model and measurements performed on conventional plaster model. Methods: The sample comprised 26 randomly selected patients to have impressions of their upper and lower jaws taken using alginate and their bite registration using a wax bite. The virtual models were obtained by scanning the alginate impression and the plaster model in a laser surface scanner (R700; 3Shape, Copenhagen, Denmark), and the measurements were performed using the Ortho Analyser (3Shape) proprietary software. The linear measurements of the size of the teeth mesial to distal, arch perimeter, intercanine distance and intermolar distance in the upper and lower arches were performed on plaster models, digital impressions and digital models, by three observers and repeated after 15 days on 8 models for intra-observer evaluations. Data were tabulated and analyzed statistically. Intra-class correlation to check the agreement of intra and inter-observers and ANOVA test were used to analyze the differences between measurements of digital models from impression and digital models from plaster. Results: The results showed a statistically significant difference (pd"0.05) for the posterior teeth, anterior teeth, upper arch perimeter and lower inter-canine distance, comparing the digital models with plaster models, but these differences are considered clinically non-significant. Conclusions: Digital models were proven be reliable and clinically acceptable for measuring tooth width, perimeter arches, intercanine and intermolar distances.

Background Erythrocytosis / polycythemia is divided into primary and secondary. Primary polycythemia can be either acquired; i.e. polycythemia vera (PV) due to somatic JAK2 mutation, or congenital due to germ-line DNA changes (erythropoietin (EPO) receptor and VHL mutations in Chuvash polycythemia). These mutations are expressed within erythroid progenitors, drive increased erythropoiesis and are detected by hypersensitive or autonomous EPO BFU-E responses. In contrast, secondary erythrocytosis (SE), such as seen with cardiopulmonary pathologies, is driven by the circulating EPO. Chronic mountain sickness (CMS) is characterized by high altitude pathological erythrocytosis and by cognitive and neurological impairments. CMS is found in subjects living in high altitude (2500 meters and higher). In La Paz, Bolivia, (3600m) there is 7% incidence of CMS erythrocytosis. Some human populations (Tibetans, Andean Quechuas and Aymaras, and Ethiopians) are adapted to very high altitudes and their adapted phenotypes and, in some instances, evolutionarily selected haplotypes, have been reported. Whole genome was evaluated in Andeans and two genes, SENP1 and ANP32D were found to be evolutionarily selected and correlated with presence or absence of erythrocytosis. The genes down-regulation in hypoxia had survival benefit in Drosophila ortholog (1).SENP1 desumoylate GATA-1 and other regulatory proteins and is critical for definitive erythropoiesis (2,3). Here we evaluated native Aymara La Paz dwellers with three types of polycythemia: CMS, SE secondary to cardiopulmonary disease, and PV, by clinical studies and by in vitro evaluation of erythroid progenitors, and compared them to non-polycythemic subjects. Patients and Methods Complete blood count was performed by automatic hematologic counter (Micro 60, USA). Serum EPO was measured by Elisa (R&D System, USA) and JAK2V617F mutation analysis by PCR assay. Erythroid progenitors were isolated by density gradient centrifugation and cultured in methylcellulose medium with and without EPO (Stem Cell technologies, Canada) at 370 C and 5 % CO2. BFU-E colonies reading was carried out according to standardized criteria at 7 and 14 days. Results Table. Normal Control(n=10) CMS (n=15) Secondary Erythrocytosis(n=10) PolycythemiaVera (n=5) 1.Gender M/F 10/0 15/0 10/0 3/2 Age (range) 42 (40-47) 48 (29-58) 53 (34-72) 67 (42-74) Hb g/dl (SD) 16.2 (+ 0.9) 20.3 (+ 0.9) 22.8 (+ 1.4) 20.0 (+ 2.5) Ret % (SD) 1.3 (+ 0.1) 2.9 (+ 1.3) 3.6 (+ 1.2) 2.1 (+ 0.3) WBC /ul (SD) 6300 (+ 1600) 7200 (+ 1900) 6600 (+ 1700) 16600 (+ 4800) PLT 103 ul (SD) 273 (+ 80) 229 (+ 58) 193 (+ 54) 604 (+ 177) sEPO mUI/ml (SD) 10.0 (+ 3.9) 10.5 (+ 2.2) 82.9 (+ 30.4) 3.0 (+ 1.2) JAK2 V617F, No. (%) 0 (0) 0 (0) 0 (0) 100 Apoptosis Normal Delayed Normal Delayed BFU-E: EEC 0 (0-0) 10 (2-25) 0 (0-0) 45 (25-70) References: 1. Yu L et al. J Exp Med., 2010, 207:1183. 2. Sharma D et al. Cell Report, 2013, 3:1640. 3. Zhou D et al. Am J Hum Genet. 2013, 93:452. 4. Kapralova K et al. Blood. 2014,123:391 Conclusions a) Endogenous erythroid colony (EEC) are present in Aymaras with CMS, indicating primary polycythemia. b) Endogenous EECs are higher in PV than in CMS. c) CMS subjects have normal serum EPO levels. d) The role of SENP1, and hypoxia-regulated RUNX1 and NF-E2 (4) that promote erythropoiesis, is being interrogated in native erythroid cells. e) It remains to be determined if the autonomous BFU-E growth is specific for Aymara's CMS or present in CMS individuals of other ethnicities. Disclosures No relevant conflicts of interest to declare.

1894 Poster Board I-917 Background Chronic Mountain Sickness (CMS) is a clinical entity that occurs in native or long-life residents above 2500 meters of altitude. The disease is characterized by massive erythrocytosis, hyperviscosity syndrome (headache, dyspnea and cyanosis) severe hypoxemia and cardiopulmonary symptoms. The etiology is unknown and no association has been found with Erythropoietin (EPO), Epo receptor (EpoR), Hypoxia Inducible Factor 1a (HIF-1a), von Hippel Lindau (VHL), as well as PHD1, PHD2, PHD3 or PTEN genes. Therapy relies on phlebotomy and oxygen support. Acetazolamide, Medroxyprogesterone and Enalapril have also been tested, but their use has not been largely implemented. Since HMG-CoA inhibitors such as farnesyltransferase inhibitors (Larghero, Blood 2005) may inhibit the in vitro autonomous erythropoiesis of polycythemia vera patients, we studied in CMS the therapeutic potential of statins that have similar pharmacologic activity. Patients and Methods Normal controls (NC, n= 10) and patients were native Bolivians from the city of La Paz, Bolivia (3600–4000 mt altitude). The diagnosis of CMS (n=15) was made according to the consensus statement on this disease (Leon-Velarde, 2005). The diagnosis of Polycythemia Vera (PV, n= 5) or secondary erythrocytosis (SE, n= 10) was done according to WHO criteria or established clinical guidelines. Serum Erythropoietin (sEpo) was assessed by chemiluminescent assay. Burst forming units-erythroid (BFU-E) assay was performed by plating 105 BM mononuclear cells in methylcellulose with or without recombinant human rhEpo (2IU/ml) and Simvastatin (20 mM). Evaluation of apoptosis by Annexin V/7-AAD and JAK2V617F mutational analyses were performed as described (Guerini et al, Leukemia 2008). Results CMS patients (median age 48 years, range 29–58) had median values of hemoglobin and hematocrit (Hb 20.3 gr/dl, range: 19.1–22 and Hct, 62%) significantly higher than observed in NC (Hb 16.2 gr/dl, range 14.8–16.5 and Htc 52%), respectively (p< 0.001) and significantly lower than SE patients (Hb: 22.8 gr/dl, range 20.2–25 and Htc: 71%), (p<0.001). Hb and Hct were not different in CMS and PV patients (p= 0.875). In CMS sEpo values (median 22 mIU/mL, range: 16.1–45.1) were significantly higher compared to NC (median 10.7 mIU/mL, range 7–18.8) (p<0.001) and lower compared to SE patients (median 82.9 mIU/mL, range 44.8–135) (p<0.001); as expected, PV patients showed very low sEpo levels (median 3 mIU/ml, range 2.5–5.2). The JAK2V617F mutation analysis proved negative in all NC, CMS and SE patients and positive in PV. In the absence of exogenous rhEpo, a median of 0, 10, 0, 45 BFU-E colonies were obtained from NC, CMS, SE and PV patients. When rhEpo was added, 21, 40, 47 and 130 BFU-E were counted, respectively; this difference was significant when comparing NC and PV to CMS (p<0.001; p< 0.001 respectively), but not in the case of SE vs. CMS (p= 0.227). Interestingly, in PV and CMS patients, BFU-E colonies remain remarkably viable between day 14–21 while viability declined rapidly in NC and SE colonies after day 14. The prolonged viability and higher sensitivity to rhEpo of BFU-E obtained from CMS and PV erythroid progenitors was also confirmed by plating BM mononuclear cells with suboptimal doses of rhEpo (0.03 to 1 IU/ml). Moreover, when simvastatin (20 μM) was added in vitro to rhEpo driven BFU-E colonies, it induced a median inhibition of 29% in NC as compared to 37, 56 and 44 in CMS, SE and PV (p <0.013: p<0.001; p<0.001, respectively). Finally, 11 CMS patients who had a concomitant hypercholesterolemia (median cholesterol level 238 mg/dl, range 206– 310) had the opportunity to be treated with statins (atorvastatin, 20–40 mg/day). Before starting treatment with atorvastatin, all patients, who had median Hb and Htc values of 19.9 gr/dl and 63 % respectively, performed phlebotomy. After a median follow up of 18 months with atorvastatin, the median Hb and Htc values were 17.1 gr/dl and 54.6 %, respectively. The need of phlebotomy was apparently reduced, from 4–6 sessions/year to 1. Conclusions Our results underline that a) hematopoietic progenitor cells from CMS patients may promote an autonomous erythroid colony growth and show hypersensitivity to hrEpo b) statins may induce in vitro a significant inhibition of this accelerated erythropoiesis so that they could play a therapeutic role in the treatment of this and other chronic myeloproliferative disorders. Disclosures: No relevant conflicts of interest to declare.

Background Polycythemia Vera (PV) is a clonal myeloproliferative neoplasm, characterized by the JAK2V617F mutation. The main goal of current therapies for PV is to prevent thrombotic events and delay transformation to Myelofibrosis (MF) or Acute Myeloid Leukemia (AML).Treatment for PV to keep an hematocrit (Hct) level <45 %, has been associated with a reduction in cardiovascular deaths and thrombotic events (Marchioli, R et al. NEJM 2013). Currently, low-risk PV patients (<60 years and no previous thrombotic events) are treated with aspirin and phlebotomy while high-risk patients require additional cytoreductive therapy, usually with Hydroxyurea (HU). Resistance to HU is associated with an increased risk of transformation and reduced survival. This is why for HU-refractory patients, second line treatments with interferon alpha, anagrelide or even ruxolitinib are recommended. In Latin America, because of high cost and drugs availability, this last group reflects difficulties to be treated. Because statins have been reported to modulate the erythroid clonogenic activity of normal BM erythroid colonies we performed a pilot study to investigate in vitro and in vivo the biologic and clinical activity of atorvastatin in PV patients Patients and Methods Ten high risk PV patients with a median age of 64.3 years (range 58-73) entered into this study. The diagnosis of PV was done according to the 2008 World Health Organization diagnostic criteria and patients were stratified according to an algorithm proposal provided by Griesshammer et al. (Ann Hematol, 2015). The definition of HU resistance (Barosi, G et al.: BJH 2009) was applicable to five patients (median age 63.9 years) failing to achieve a satisfactory hematologic response upon treatment with more than 2 g of HU, 100 mg of Aspirin and phlebotomies. The assessment of the JAK2V617F mutation was performed as previously described (Guerini et al.: Leukemia 2009). Colony assay, proliferation and apoptosis tests were performed with or without Simvastatin (3.5 uM), as previously described (Amaru, A, Experimental Hematology 2012), on cell lines (UKE1 and K562) and bone marrow mononuclear cells obtained from PV patients and healthy donors. Patients with HU refractory PV (n=5) and high risk PV with hypercholesterolemia (n=5) were eligible to receive Atorvastatin (20 mg/day) added on the top of the ongoing treatment with phlebotomies, Aspirin (100 mg/day) and cytoreductive HU therapy (500 mg/day). All treated patients were high altitude residents (> 3.600 m.a.s.l.) of La Paz (Bolivia) where the normal Hct level of healthy subjects is 48-57% for men and 44-54% for women. This pilot study was approved by the Review Board of the Hospital and the University of San Andres, La Paz. Results In a preliminary set of in vitro proliferation cell assays, simvastatin (3.5 uM), added for 5 days, induced a 33% inhibition of cell proliferation of UKE-1 (JAK2V617F mutated) as compared to 5 % of K562 (BCR/ABL positive). A comparable result was obtained in a 7-day clonogenic cell assay where the colony inhibition was 50 % for UKE-1 and 10 % for K562. On the basis of these results similar experiments were also performed using BM mononuclear cells derived from PV patients and healthy donors. In these experiments performed with the addition of simvastatin, it induced a 41% of inhibition in BFU-E colonies of PV patients and a 25% of inhibition in healthy donors. Furthermore, BFU-E colonies inhibited by simvastatin presented a decrease in hemoglobinization and the size of colonies. HU refractory PV patients and High-risk PV patients with hypercholesterolemia treated with the addition of Atorvastatin, Aspirin, cytoreductive HU and phlebotomies; after a follow-up of 2.6 years (1-7 years), induced a decrease of WBC from 16.500 to 9.270/ul, Hct 61.1 to 52.3% and PLT 457.900,000 to 324.7000/ul. The number of required phlebotomies is reduced in comparison to the required at starting treatment. None of the patients presented thrombotic or cardiopulmonary event. One patient died within two years of starting treatment, due to complications of diabetes mellitus. Conclusions In vitro and in vivo, statins showed some evidence of inhibitory activity of the hematopoiesis of PV patients. These preliminary results might indicate the opportunity to further investigate the potential clinical value of these molecules in the treatment of PV. Disclosures Off Label Use: Atorvastatin was used for its antiproliferative activity on myeloid progenitor cells shown by in vitro experiments.

Dedico este trabalho a Deus, pоr ser essencial еm minha vida, y a mis mayores tesoros, Toribio y Elsa, que me dieron fuerza y coraje en toda esta caminada, renunciando a muchas cosas y sueños para poder realizar los míos. AGRADECIMENTOS Agradecimentos aos ProfessoresÀ Faculdade de Odontologia de Piracicaba, na pessoa do seu diretor Prof. Dr. Guilherme Elias Pessanha Henriques, por ter me oferecido a oportunidade de conhecer e conviver com profissionais renomados em suas áreas.Ao meu querido orientador Prof. Dr. Frab Norberto Bóscolo, obrigada por ter me ensinado a enxergar o outro lado da moeda, por me direcionar, por subsidiar minha coragem de querer sempre lutar pelo que mais quero, por dividir seu tempo, paciência e amizade. Sou uma pessoa privilegiada por ter um professor tão competente em todos os aspectos ao meu lado! Foi uma honra ter sido sua orientada! ...E mais ainda obrigada pelo carinho de paizinho que sempre me mostrou! À minha querida mãezinha brasileira Profª. Dra. Solange Maria de Almeida, que sempre foi muito mais que uma professora, é uma amiga, mãe e conselheira que me mostrou a importância de me questionar, duvidar, pensar e sonhar. Hoje cheguei aqui através do seu incentivo e confiança em mim. Obrigada! Ao Prof. Dr. Francisco Haiter Neto, grande mestre, agradeço por tudo o que me ensinou e me proporcionou a aprender. Mesmo com tantos afazeres sempre esteve à disposição para tirar dúvidas, conversar sobre os trabalhos e me ajudar de forma exemplar quando precisava. Obrigada pelo carinho e atenção! À Profª. Dra. Deborah Queiroz de Freitas, pela sua dedicação e competência. Obrigada por estar à nossa disposição mesmo sempre muito ocupada! Ao Prof. Dr. Matheus Oliveira pessoa que admiro pelo profissionalismo e competência, agradeço também pelos conselhos! Ao Prof. Dr. Francisco Carlos Groppo, pela atenção, carinho e amizade! Obrigada pelos conselhos, ensinamentos e a delicadeza ao me receber em sua sala! Você não imagina como fico feliz em ter tido contato com o senhor na realização deste trabalho! Aos professores membros da banca do exame de qualificação, Prof. Dr.