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In this paper the osteobiography of an elderly woman recovered from a cemetery tomb where she was buried in 1850, affected by hip fracture and osteoporosis, is described. The overall anthropological characteristics of the individual have been investigated. Macroscopic, radiographic, tomographic, microscopic, and chemical and structural examinations have been performed to give a detailed account of the condition of the skeleton. A non-union pertrochanteric fracture not surgically treated and probably due to senile osteoporosis was diagnosed. The consequences of the fracture to the bones show that this individual likely survived several years following the injury. The osseous features we describe (remodelled bone at the fracture site, asymmetry of entheseal changes likely related to the particular walking pattern of the individual) may be useful in personal identification of skeletons of legal interest. Regarding the recognition of osteoporosis in unearthed skeletons, our study underlines that the cortical thickness, microscopic features, degree of crystallinity and Ca/P ratio represent more useful elements than the mean bone density, mineral/matrix ratio and mineral maturity, which are more sensitive to diagenetic changes that affect the mineral phase post-mortem.

Polymerized resin-based materials are successfully utilized in medical applications. One drawback is the release of monomers from the matrix due to an incomplete polymerization or degradation processes. Released monomers can diffuse in the systemic circulation and induce adverse effects to biological tissues. Although there are many hypotheses about the induction of cell death by resin monomers, the underlying mechanisms are still under discussion. The aim of the study was to investigate the morphological modifications in human gingival fibroblasts exposed to 2-hydroxy-ethyl methacrylate (HEMA) to better elucidate the mechanism of cell death induced by resin monomers. Primary cultures of gingival fibroblasts were exposed to 3mM HEMA for 24 h, 72 h, 96 h. Morphological investigations were performed by scanning and transmission electron microscopy, while western blot for caspase-3 was carried out to verify apoptosis. Electron microscopy images showed deep changes in the cell surface and cytoplasm after 72 h and 96 h of HEMA treatment. Autophagic vesicles were easily observed just after 24 h. Cleaved caspase-3 was detected after 72 h of treatment. These findings suggest that resin based materials induced cell death by the cooperation of apoptosis and autophagy mechanisms. The understanding of these mechanisms will lead to the development of smart biomaterials without or with low adverse effects., Italian Journal of Anatomy and Embryology, Vol 119, No 2 (2014)

Up regulation of matricellular proteins are often involved in in-vitro cell suffering and in in-vivo encapsulation, foreign body reaction and inflammation response following scaffold implantation [1]. The cytotoxicity of biomaterials is generally tested according to the ISO standard 10993-5, based mainly on viability tests. Further assays, based on the improved biocompatibility knowledge, could be suggested to better analyze the cytotoxicity of implant materials. The aim of the study was to propose matricellular protein expression as a new assay in the evaluation of cytotoxicity of implant materials. Tenascin C, osteocalcin and osteopontin belonged to the matricellular protein family and they were chosen as cytotoxicity markers. Viability test, Real Time PCR and western blot in mesenchymal stem cells seeded on collagen/hydroxyapatite scaffold were performed to evaluate gene/protein expression. Transmission electron microscopy was carried out to evaluate morphological changes induced by cell/scaffold interactions. Results showed an high viability of the cells during the time of culture and a good cell adhesion and morphology observed by electron microscopy. A lower expression of tenascin-c, osteonectin and osteopontin compared to cells cultured on tissue flasks was demonstrated both by Real Time PCR and western blot suggesting a better cell culture condition in 3D biomimetic scaffold compared to tissue flasks. Based on our results, we propose the analysis of matricellular protein expression as a new parameter for testing cytotoxicity of implant materials., Italian Journal of Anatomy and Embryology, Vol 118, No 2 (Supplement) 2013

Background: Deletions of IKAROS (IKZF1) frequently occur in B-cell precursor acute lymphoblastic leukemia (B-ALL) but the mechanisms by which they influence pathogenesis are unclear. To address this issue, a cohort of 144 adult B-ALL patients (106 BCR-ABL1-positive and 38 B-ALL negative for known molecular rearrangements) was screened for IKZF1 deletions by single nucleotide polymorphism (SNP) arrays; a sub-cohort of these patients (44%) was then analyzed for gene expression profiling. Principal Findings: Total or partial deletions of IKZF1 were more frequent in BCR-ABL1-positive than in BCR-ABL1-negative B-ALL cases (75% vs 58%, respectively, p = 0.04). Comparison of the gene expression signatures of patients carrying IKZF1 deletion vs those without showed a unique signature featured by down-regulation of B-cell lineage and DNA repair genes and up-regulation of genes involved in cell cycle, JAK-STAT signalling and stem cell self-renewal. Through chromatin immunoprecipitation and luciferase reporter assays we corroborated these findings both in vivo and in vitro, showing that Ikaros deleted isoforms lacked the ability to directly regulate a large group of the genes in the signature, such as IGLL1, BLK, EBF1, MSH2, BUB3, ETV6, YES1, CDKN1A (p21), CDKN2C (p18) and MCL1. Conclusions: Here we identified and validated for the first time molecular pathways specifically controlled by IKZF1, shedding light into IKZF1 role in B-ALL pathogenesis.

Background Measurements of plantar loading reveal foot-to-floor interaction during activity, but information on bone architecture cannot be derived. Recently, cone-beam computer tomography (CBCT) has given visual access to skeletal structures in weight-bearing. The combination of the two measures has the potential to improve clinical understanding and prevention of diabetic foot ulcers. This study explores the correlations between static 3D bone alignment and dynamic plantar loading. Methods Sixteen patients with diabetes were enrolled (group ALL): 15 type 1 with (N, 7) and without (D, 8) diabetic neuropathy, and 1 with latent autoimmune diabetes. CBCT foot scans were taken in single-leg upright posture. 3D bone models were obtained by image segmentation and aligned in a foot anatomical reference frame. Absolute inclination and relative orientation angles and heights of the bones were calculated. Pressure patterns were also acquired during barefoot level walking at self-selected speed, from which regional peak pressure and absolute and normalised pressure-time integral were worked out at hallux and at first, central and fifth metatarsals (LOAD variables) as averaged over five trials. Correlations with 3D alignments were searched also with arch index, contact time, age, BMI, years of disease and a neuropathy-related variable. Results Lateral and 3D angles showed the highest percentage of significant (p Conclusion These preliminary original measures reveal that alteration of plantar dynamic loading patterns can be accounted for peculiar structural changes of foot bones. Load under the central metatarsal heads were correlated more with inclination of the corresponding phalanxes than metatarsals. Further analyses shall detect to which extent variables play a role in the many group-specific correlations.

Purpose To compare weight-bearing cone-beam computer tomography (CBCT) and conventional computer tomography (CT)-based measurements of patellofemoral alignment and stability in patients surgically treated for recurrent patellar dislocation. These scans implied respectively single-leg up-right posture, the knee flexed, and lower limb muscles activation, versus supine position with the knee extended. Methods A total of 17 patients (11 males/6 females) after surgical reconstruction with fascia lata allograft for recurrent patellofemoral dislocation were analyzed at 60-month follow-up. Tilt and congruence angles and tibial tuberosity–trochlear groove (TT-TG) offset were measured on images obtained from CBCT and conventional CT scans by three independent and expert radiologists. Paired t tests were performed to compare measurements obtained from the two scans. Inter-rater reliability was assessed using a two-way mixed-effects model intra-class correlation coefficient (ICC). Results Only TT-TG offset was found significantly smaller (p Conclusion In patients surgically treated for recurrent patellar dislocation, TT-TG offset was found overestimated with conventional CT. All measurements of patellofemoral stability and alignment were found more consistent when obtained with weight-bearing CBCT compared to conventional CT.

Despite the genomic characterization of pancreatic cancer (PC), marked advances in the development of prognosis classification and novel therapeutic strategies have yet to come. The present study aimed to better understand the genomic alterations associated with the invasive phenotype of PC, in order to improve patient selection for treatment options. A total of 30 PC samples were analysed by either whole transcriptome (9 samples) or exome sequencing (21 samples) on an Illumina platform (75X2 or 100X2 bp), and the results were matched with normal DNA to identify somatic events. Single nucleotide variants and insertions and deletions were annotated using public databases, and the pathogenicity of the identified variants was defined according to prior knowledge and mutation-prediction tools. A total of 43 recurrently altered genes were identified, which were involved in numerous pathways, including chromatin remodelling and DNA damage repair. In addition, an analysis limited to a subgroup of early stage patients (50% of samples) demonstrated that poor prognosis was significantly associated with a higher number of known PC mutations (P=0.047). Samples from patients with a better overall survival (>25 months) harboured an average of 24 events, whereas samples from patients with an overall survival of

Novel polylactide (PLA) microspheres endowed with hydrophilic and bioadhesive surfaces as injectable formulations for the controlled release of fenretinide were prepared, using a novel technique based on the co-precipitation of PLA with gelatin, at the interface of a liquid dispersion formed by the addition of N-methylpyrrolidone containing PLA and dextrin (DX), towards an aqueous solution of gelatin (G). The resulting PLA-G-DX microspheres were compared with others prepared by the same technique using polylactide-co-glycolide (PLGA), with or without DX, and with or without phosphatidylcholine. Of the different systems, the PLA-G-DX microspheres had the best morphological, dimensional and functional characteristics. They had the highest drug loading, and their drug release was the most efficient over time without any burst effect. Their in vitro anti-tumoural activity was strongly enhanced with respect to the pure fenretinide. This paralleled the increased drug concentration inside the cells due to their marked bioadhesion to the tumour cell membranes as indicated by scanning electron microscope images.

BACKGROUND: The role of the microenvironment during the initiation and progression of carcinogenesis is thought to be of critical importance, both for the enhanced understanding of fundamental cancer biology as well as for improving molecular diagnostics and therapeutics. The aim of this study was to establish an in vitro model based on a co-culture of healthy human fibroblasts (HFs) and human osteosarcoma cells (MG-63s) to simulate the microenvironment including tumor and healthy cells. METHODS: The HFs and MG-63s were in vitro co-cultured for a period of time ranging from 24 h to 7 days. Cell morphology and organization were studied using phase contrast microscopy while the expression of Human Cartilage Glycoprotein 39 (YKL-40), Vascular Endothelial Growth Factor (VEGF) and Matrix Metalloprotease 1 (MMP1) was investigated by Real Time PCR and Western Blotting. RESULTS: The results showed a characteristic disposition of tumor and healthy co-cultured cells in columns which are not visible in tumor and healthy cells grown singularly. The expression of YKL-40, VEGF and MMP1 significantly changed in co-cultured cells compared to HFs and MG-63s separately cultured. CONCLUSIONS: We concluded that the tumor microenvironment has an influence on the protein expression of the healthy surrounding tissues and the process of tumorigenicity.

Abstract 3935 Background The p53 tumor suppressor pathway is tightly kept in check, or completely silenced in cancer cells. A potent inhibitor of p53 is represented by MDM4, which is critical for the control of p53 activity during the response to stress and is often amplified in several types of cancer. TP53 mutations are rare in newly diagnosed MM, while occur more frequently as late event in the course of the disease and are related to survival. Recently, the adverse prognostic impact of chr. 1q amplification, described in almost 40% of newly diagnosed MM pts, has been reported. The minimal amplified region on chr. 1q harbors MDM4. Since both del(17p) and amp(1q) identify a subgroup of high-risk MM pts, even when the novel agents are part of up-front treatment strategy, we molecularly analyzed a subgroup of MM patients treated with bortezomib-thalidomide-dexamethasone (VTD) incorporated into autologous stem cell transplantation, in order to investigate mechanisms which might be activated in myeloma plasma cells to direct and/or indirect limit the p53 function. Methods Thirty eight pts treated with VTD incorporated into autologous stem cell transplantation were analysed by means of gene expression profile (Affymetrix U133 Plus2.0 array) and unpaired analysis of copy number alterations (CNA) (Affymetrix 6.0 SNP array). Both GEP and SNP arrays experiments were performed on highly purified CD138+ bone marrow plasma cells obtained at diagnosis from each pts. The presence of CNAs in chr.1 and 17 was evaluated to identify pts carrying amp(1q) and del(17p). Results Eighteen out of 38 pts (42%) carried a minimal amplification region of 1,1 Mb on chr.1q, which harbors MDM4. Five out of 38 pts (13%) carried a minimal deletion region of 482 Kb on chr.17, which harbors TP53. To explore the involvement of the p53 pathway in MM, pts were stratified according to the presence of amplified MDM4 and/or deleted p53 (group A, 18 pts) or the absence of both these abnormalities (group B, 20 pts). Baseline clinical characteristics were homogeneous, except for a higher rate of ≥ 3 bone lesions in pts carrying amplified MDM4 and/or deleted p53. The rate of best complete or near complete response was 89% in group A and 75% in group B. With a median follow-up of 36 months, the risk of relapse or progression was 50% for pts in group A and 25% for those in group B. The average number of aberrations per group was overall higher in group A as compared to group B (165 vs. 103 CNAs, p =0.03); indeed, the presence of amplified MDM4 and/or deleted p53 was significantly associated with a list of 95 CNAs (clustered on chr. 1, 2, 6, 8, 11, 13, 16 and 18), which included del16q (with a minimal area of deletion including WWOX), observed in 39% vs. 5% cases from group A and B, respectively (p Conclusions Pts carrying amplified MDM4 and/or deleted p53 showed a significantly higher number of CNAs and the significant over-expression of genes involved in the response mechanisms to genotoxic stress, as compared to pts lacking these chromosomal aberrations. This might account for the worse outcome of patients harboring del(17p) and/or amp(1q). The amplification of MDM4 locus and the over-expression of YY1 might contribute to maintain p53 in an OFF state by an indirect mechanism. Additional data on the role of both direct and indirect control of p53 pathway on VTD-treated MM pts prognosis, extended to an higher number of pts, will be presented during the meeting. Supported by: Fondazione Del Monte di Bo e Ra, Ateneo RFO grants (M.C.) BolognAIL. Disclosures: Cavo: Genzyme: Honoraria.

Background. Efficacy of Thalidomide-dexamethasone (TD) as induction therapy in preparation for autologous stem cell transplantation (ASCT) in multiple myeloma provided the basis for the design of the phase II “Bologna 2002”study incorporating TD into double autotransplantation as up-front therapy for younger patients (pts) with newly diagnosed disease. Aim. We performed a molecular study aimed at identifying a gene expression profile (GEP) signature predictive of attainment of at least near complete response (nCR) to TD and subsequent autotransplantation. Methods. For this purpose, we analyzed bone marrow samples obtained at diagnosis from 112 pts who received TD before double ASCT. The differential gene expression of CD138+ enriched plasma cells was evaluated by means of expression microarray using the Affymetrix platform. Significant expression results were validated by Real-time PCR. Results. Two subsequent study phases were planned. Firstly, a GEP supervised analysis was performed on a training set of 32 pts, allowing to identify 157 probe sets differentially expressed (P

// Sandra Durante 1,* , Isabella Orienti 2,* , Gabriella Teti 1 , Viviana Salvatore 1 , Stefano Focaroli 1 , Anna Tesei 3 , Sara Pignatta 3 and Mirella Falconi 1 1 DIBINEM—Department of Biomedical and Neuromotor Sciences, University of Bologna, Via Irnerio 48, 40126, Bologna, Italy 2 FaBiT–Department of Pharmacy and Biotechnology, University of Bologna, Via San Donato 19, 240127, Bologna, Italy 3 Biosciences Laboratory, Istituto Scientifico Romagnolo per lo Studio e la Cura dei Tumori(IRST) IRCCS, Biosciences Laboratory, via P. Maroncelli 40, 47014, Meldola, FC, Italy * These authors contributed equally to this work Correspondence: Mirella Falconi, email: // Keywords : Received : May 7, 2014 Accepted : May 28, 2014 Published : May 28, 2014 Abstract Sufficient knowledge regarding cellular and molecular basis of lung cancer progression and metastasis would help in the development of novel and effective strategies for the treatment of lung cancer. 4HPR is a synthetic retinoid with potential anti-tumor activity but is still limited because of its poor bioavailability. The use of albumin as a complexing agent for a hydrophobic drug is expected to improve the water solubility and consequently their bioavailability.This study investigated the antitumor activity of a novel complex between albumin and 4-HPR in a mouse model of human lung cancer and focuses on role and mechanism of Cav-1 mainly involved in regulating cancer and Acsvl3 mainly connected with tumor growth. Their expressions were assayed by immunohistochemistry and qRT-PCR, to demonstrate the reduction of the tumor growth following the drug treatment. Our results showed a high antitumor activity of 4HPR-HSA by reduction of the volume of tumor mass and the presence of a high level of apoptotic cell by TUNEL assay. The downregulation of Cav-1 and Acsvl3 suggested a reduction of tumor growth. In conclusion, we demonstrated the great potential of 4HPR-HSA in the treatment of lung cancer. More data about the mechanism of drug delivery the 4HPR-HSA are necessary.

Abstract 2901 Hypoxia-inducible factor-1 alpha (HIF1 α) is a transcription factor that plays a critical role in survival and angiogenesis. In solid tumors, elevated expression of HIF-1 α, in response to hypoxia or activation of growth factor pathways, is associated with tumor proliferation, metastasis, and drug resistance and correlated with poor prognosis. In contrast to solid tumors, the role of HIF1 α in hematological malignancies is not completely known. In particular in multiple myeloma (MM) HIF1 α has been suggested to be constitutively expressed and HIF1 α knockdown cell lines have shown higher sensitivity to standard chemotherapy, suggesting a role in the pathophysiology of MM. In the present study, we explored the effect of EZN2968, an antisense oligonucleotide against HIF1 α, as a molecular target in MM. We showed, using real time PCR, and Western blotting analysis, that the expression of HIF1 α in several MM cell lines (MM1S, U266, OPM2, RPMI8226) is detectable under conditions of normoxia or hypoxia and is increased in the presence of growth stimuli (IL-6 and stroma cells). The immunofluorescence analysis suggested that the protein is ubiquitously present in both the cytosol and nucleus. To evaluate the specificity of the oligonucleotide for the target, we tested whether EZN2968 was able to induce a selective and stable down-modulation of HIF1 α mRNA and protein expression. We confirmed that the downmodulation was lasting in a long term culture experiment (up to 96 hours) either in normoxic or hypoxic conditions, and did not affect the expression of other family members of hypoxia inducible transcription factors (HIF2 α). We next explored the effects of EZN-2968 on the growth and survival of MM cells. Using an MTT colorimetric survival assay, we showed that, after 48 hours of culture in the presence of the HIF1 α inhibitor (20μM), MM1.S and U266 cell lines exhibited a reduction of 30% of viability compared to untreated cells, while RPMI8226 of 15%. AnnexinV/PI staining revealed that EZN-2968 (20μM) increased, after 48 hours of culture, the percentage of PI+ cells compared to the control, suggesting a disruption on membrane permeability. In addition, immunoblotting revealed PARP cleavage as early as 24 hours. Evaluation of cell cycle profile, by flow cytometric analysis, showed an increase of the sub-G0/G1 population from 3.5% to 30 %, after 48 hour of exposure to EZN-2968. To evaluate if the impact on cell viability was irreversible, we performed a cell death commitment assays. MM1S cells were incubated with EZN2968 (20 μM) for 24 to 96 hours, following incubation in drug-free medium for additional 24 to 72 hours. MTT colorimetric survival assay showed that EZN-2968 treatment for as early as 24h resulted in commitment to death in all cell lines tested. To evaluate the effect of microenvironment, MM cells treated with EZN2968 were exposed to IL-6 and stroma cells for additional 24 hours. EZN2968 overcame the proliferative effect induced by cytokines. We next evaluated the impact of EZN-2968 on purified CD138+ cells from MM patients with advanced MM. MTT colorimetric survival assay showed a reduction of cells viability of 30% after 24 hours of incubation. In addition we observed a low sensitivity of PBMCs and CD34+cells, derived from healthy donors, to EZN-2968 treatment suggesting that EZN-2968 has selective in vitro activity against MM cells. Evaluation of gene expression profiling modulation induced by EZN 2968 is on going. In summary, our results suggests that the inhibition of HIF1 α activity can be used as an attractive therapeutic target for MM patients and provide the rationale for clinical evaluation of HIF inhibitors. Disclosures: No relevant conflicts of interest to declare.