Combined forced diuresis and late acquisition on [68Ga]Ga-PSMA-11 PET/CT for biochemical recurrent prostate cancer: a clinical practice-oriented study.

OBJECTIVES: Increased detection of prostate cancer (PCa) recurrences using [68Ga]Ga-PSMA-11 PET/CT has been reported by adding forced diuresis or late-phase imaging to the standard protocol. However, the combination of these procedures in the clinical setting is still not standardized. METHODS: One hundred prospectively recruited biochemical recurrent PCa patients were restaged with dual-phase [68Ga]Ga-PSMA-11 PET/CT from September 2020 to October 2021. All patients received a standard scan (60 min), followed by diuretics (140 min) and a late-phase abdominopelvic scan (180 min). PET readers with low (n = 2), intermediate (n = 2), or high (n = 2) experience rated (i) standard and (ii) standard + forced diuresis late-phase images in a stepwise fashion according to E-PSMA guidelines, scoring their level of confidence. Study endpoints were (i) accuracy against a composite reference standard, (ii) reader's confidence level, and (iii) interobserver agreement. RESULTS: Forced diuresis late-phase imaging increased the reader's confidence category for local and nodal restaging (both p < 0.0001), and the interobserver agreement in identifying nodal recurrences (from moderate to substantial, p < 0.01). However, it significantly increased diagnostic accuracy exclusively for local uptakes rated by low-experienced readers (from 76.5 to 84%, p = 0.05) and for nodal uptakes rated as uncertain at standard imaging (from 68.1 to 78.5%, p < 0.05). In this framework, SUVmax kinetics resulted in an independent predictor of PCa recurrence compared to standard metrics, potentially guiding the dual-phase PET/CT interpretation. CONCLUSIONS: The present results do not support the systematic combination of forced diuresis and late-phase imaging in the clinical setting, but allow the identification of patients-, lesions-, and reader-based scenarios that might benefit from it. KEY POINTS: • Increased detection of prostate cancer recurrences has been reported by adding diuretics administration or an additional late abdominopelvic scan to the standard [68Ga]Ga-PSMA-11 PET/CT procedure. • We verified the added value of combined forced diuresis and delayed imaging, showing that this protocol only slightly increases the diagnostic accuracy of [68Ga]Ga-PSMA-11 PET/CT, thus not justifying its systematic use in clinics. • However, it can be helpful in specific clinical scenarios, e.g., when PET/CT is reported by low-experienced readers. Moreover, it increased the reader's confidence and the agreement among observers.

The P2X7 purinergic receptor in intervertebral disc degeneration.

Mechanisms involved in the development of intervertebral disc (IVD) degeneration are only partially known, thus making the implementation of effective therapies very difficult. In this study, we investigated P2X7 purinergic receptor (P2X7R), NLRP3 inflammasome, and interleukin (IL)-1ß expression in IVD specimens at different stages of disease progression, and during the in vitro dedifferentiation process of the primary cells derived thereof. We found that P2X7R, NLRP3, and IL-1ß expression was higher in the IVD samples at a more advanced stage of degeneration and in the expanded IVD cells in culture which partially recapitulated the in vivo degeneration process. In IVD cells, the P2X7R showed a striking nuclear localization, while NLRP3 was mainly cytoplasmic. Stimulation with the semiselective P2X7R agonist benzoyl ATP together with lipopolysaccharide treatment triggered P2X7R transfer to the cytoplasm and P2X7R/NLRP3 colocalization. Taken together, these findings support pathophysiological evidence that the degenerated disc is a highly inflamed microenvironment and highlight the P2X7R/NLRP3 axis as a suitable therapeutic target. The immunohistochemical analysis and the assessment of subcellular localization revealed a substantial expression of P2X7R also in normal disc tissue. This gives us the opportunity to contribute to the few studies performed in natively expressed human P2X7R so far, and to understand the possible physiological ATP-mediated P2X7R homeostasis signaling. Therefore, collectively, our findings may offer a new perspective and pave the way for the exploration of a role of P2X7R-mediated purinergic signaling in IVD metabolism that goes beyond its involvement in inflammation.

Expression and function of the P2X7 receptor in human osteoblasts: The role of NFATc1 transcription factor.

Bone mineralization is an orchestrated process by which mineral crystals are deposited by osteoblasts; however, the detailed mechanisms remain to be elucidated. The presence of P2X7 receptor (P2X7R) in immature and mature bone cells is well established, but contrasting evidence on its role in osteogenic differentiation and deposition of calcified bone matrix remains. To clarify these controversies in the present study, we investigated P2X7R participation in bone maturation. We demonstrated that the P2X7R is expressed and functional in human primary osteoblasts, and identified in the P2RX7 promoter several binding sites for transcription factors involved in bone mineralization. Of particular interest was the finding that P2X7R expression is enhanced by nuclear factor of activated T cells cytoplasmic 1 (NFATc1) overexpression, and accordingly, NFATc1 is recruited at the P2RX7 gene promoter in SaOS2 osteoblastic-like cells. In conclusion, our data provide further insights into the regulation of P2X7R expression and support the development of drugs targeting this receptor for the therapy of bone diseases.

Ectopic expression of PLC-ß2 in non-invasive breast tumor cells plays a protective role against malignant progression and is correlated with the deregulation of miR-146a.

Cells in non-invasive breast lesions are widely believed to possess molecular alterations that render them either susceptible or refractory to the acquisition of invasive capability. One such alteration could be the ectopic expression of the ß2 isoform of phosphoinositide-dependent phospholipase C (PLC-ß2), known to counteract the effects of hypoxia in low-invasive breast tumor-derived cells. Here, we studied the correlation between PLC-ß2 levels and the propensity of non-invasive breast tumor cells to acquire malignant features. Using archival FFPE samples and DCIS-derived cells, we demonstrate that PLC-ß2 is up-regulated in DCIS and that its forced down-modulation induces an epithelial-to-mesenchymal shift, expression of the cancer stem cell marker CD133, and the acquisition of invasive properties. The ectopic expression of PLC-ß2 in non-transformed and DCIS-derived cells is, to some extent, dependent on the de-regulation of miR-146a, a tumor suppressor miRNA in invasive breast cancer. Interestingly, an inverse relationship between the two molecules, indicative of a role of miR-146a in targeting PLC-ß2, was not detected in primary DCIS from patients who developed a second invasive breast neoplasia. This suggests that alterations of the PLC-ß2/miR-146a relationship in DCIS may constitute a molecular risk factor for the appearance of new breast lesions. Since neither traditional classification systems nor molecular characterizations are able to predict the malignant potential of DCIS, as is possible for invasive ductal carcinoma (IDC), we propose that the assessment of the PLC-ß2/miR-146a levels at diagnosis could be beneficial for identifying whether DCIS patients may have either a low or high propensity for invasive recurrence.

Interplay between spinal cord and cerebral cortex metabolism in amyotrophic lateral sclerosis.

We recently reported the potential of Hough transform in delineating spinal cord metabolism by 18F-fluorodeoxyglucose PET/CT scanning in amyotrophic lateral sclerosis. The present study aimed to verify the relationship between spinal cord and brain metabolism in 44 prospectively recruited patients affected by amyotrophic lateral sclerosis submitted to 18F-fluorodeoxyglucose brain and whole-body PET/CT. Patients were studied to highlight the presence of brain hypo- or hypermetabolism with respect to healthy controls, and multiple regression analysis was performed to evaluate the correlation between spinal cord and brain metabolism. Our results confirmed higher 18F-fluorodeoxyglucose uptake in both cervical and dorsal spinal cord in patients with amyotrophic lateral sclerosis with respect to controls. This finding was paralleled by the opposite pattern in the brain cortex that showed a generalized reduction in tracer uptake. This hypometabolism was particularly evident in wide regions of the frontal-dorsolateral cortex while it did not involve the midbrain. Bulbar and spinal disease onset was associated with similar degree of metabolic activation in the spinal cord. However, among spinal onset patients, upper limb presentation was associated with a more pronounced metabolic activation of cervical segment. Obtained data suggest a differential neuro-pathological state or temporal sequence in disease progression.

"Bridging the Gap" Everything that Could Have Been Avoided If We Had Applied Gender Medicine, Pharmacogenetics and Personalized Medicine in the Gender-Omics and Sex-Omics Era.

. Gender medicine is the first step of personalized medicine and patient-centred care, an essential development to achieve the standard goal of a holistic approach to patients and diseases. By addressing the interrelation and integration of biological markers (i.e., sex) with indicators of psychological/cultural behaviour (i.e., gender), gender medicine represents the crucial assumption for achieving the personalized health-care required in the third millennium. However, 'sex' and 'gender' are often misused as synonyms, leading to frequent misunderstandings in those who are not deeply involved in the field. Overall, we have to face the evidence that biological, genetic, epigenetic, psycho-social, cultural, and environmental factors mutually interact in defining sex/gender differences, and at the same time in establishing potential unwanted sex/gender disparities. Prioritizing the role of sex/gender in physiological and pathological processes is crucial in terms of efficient prevention, clinical signs' identification, prognosis definition, and therapy optimization. In this regard, the omics-approach has become a powerful tool to identify sex/gender-specific disease markers, with potential benefits also in terms of socio-psychological wellbeing for each individual, and cost-effectiveness for National Healthcare systems. "Being a male or being a female" is indeed important from a health point of view and it is no longer possible to avoid "sex and gender lens" when approaching patients. Accordingly, personalized healthcare must be based on evidence from targeted research studies aimed at understanding how sex and gender influence health across the entire life span. The rapid development of genetic tools in the molecular medicine approaches and their impact in healthcare is an example of highly specialized applications that have moved from specialists to primary care providers (e.g., pharmacogenetic and pharmacogenomic applications in routine medical practice). Gender medicine needs to follow the same path and become an established medical approach. To face the genetic, molecular and pharmacological bases of the existing sex/gender gap by means of omics approaches will pave the way to the discovery and identification of novel drug-targets/therapeutic protocols, personalized laboratory tests and diagnostic procedures (sex/gender-omics). In this scenario, the aim of the present review is not to simply resume the state-of-the-art in the field, rather an opportunity to gain insights into gender medicine, spanning from molecular up to social and psychological stances. The description and critical discussion of some key selected multidisciplinary topics considered as paradigmatic of sex/gender differences and sex/gender inequalities will allow to draft and design strategies useful to fill the existing gap and move forward.

Vav1 is necessary for PU.1 mediated upmodulation of miR-29b in acute myeloid leukaemia-derived cells.

It has been recently demonstrated that high pre-treatment levels of miR-29b positively correlated with the response of patients with acute myeloid leukaemia (AML) to hypomethylating agents. Upmodulation of miR-29b by restoring its transcriptional machinery appears indeed a tool to improve therapeutic response in AML. In cells from acute promyelocytic leukaemia (APL), miR-29b is regulated by PU.1, in turn upmodulated by agonists currently used to treat APL. We explored here the ability of PU.1 to also regulate miR-29b in non-APL cells, in order to identify agonists that, upmodulating PU.1 may be beneficial in hypomethylating agents-based therapies. We found that PU.1 may regulate miR-29b in the non-APL Kasumi-1 cells, showing the t(8;21) chromosomal rearrangement, which is prevalent in AML and correlated with a relatively low survival. We demonstrated that the PU.1-mediated contribution of the 2 miR-29b precursors is cell-related and almost completely dependent on adequate levels of Vav1. Nuclear PU.1/Vav1 association accompanies the transcription of miR-29b but, at variance with the APL-derived NB4 cells, in which the protein is required for the association of PU.1 with both miRNA promoters, Vav1 is part of molecular complexes to the PU.1 consensus site in Kasumi-1. Our results add new information on the transcriptional machinery that regulates miR-29b expression in AML-derived cells and may help in identifying drugs useful in upmodulation of this miRNA in pre-treatment of patients with non-APL leukaemia who can take advantage from hypomethylating agent-based therapies.

Hypoxia Preconditioning of Human MSCs: a Direct Evidence of HIF-1α and Collagen Type XV Correlation.

BACKGROUND/AIMS: Mesenchymal stromal cells (MSCs) hold considerable promise in bone tissue engineering, but their poor survival and potency when in vivo implanted limits their therapeutic potential. For this reason, the study on culture conditions and cellular signals that can influence the potential therapeutic outcomes of MSCs have received considerable attention in recent years. Cell maintenance under hypoxic conditions, in particular for a short period, is beneficial for MSCs, as low O2 tension is similar to that present in the physiologic niche, however the precise mechanism through which hypoxia preconditioning affects these cells remains unclear. METHODS: In order to explore what happens during the first 48 h of hypoxia preconditioning in human MSCs (hMSCs) from bone marrow, the cells were exposed to 1.5% O2 tension in the X3 Hypoxia Hood and Culture Combo - Xvivo System device. The expression modulation of critical genes which could be good markers of increased osteopotency has been investigated by Western blot, immunufluorescence and ELISA. Luciferase reporter assay and Chromatin immunoprecipitation was used to investigate the regulation of the expression of Collagen type XV (ColXV) gene. RESULTS: We identified ColXV as a new low O2 tension sensitive gene, and provided a novel mechanistic evidence that directly HIF-1α (hypoxia-inducible factor-1 alpha) mediates ColXV expression in response to hypoxia, since it was found specifically in vivo recruited at ColXV promoter, in hypoxia-preconditioned hMSCs. This finding, together the evidence that also Runx2, VEGF and FGF-2 expression increased in hypoxia preconditioned hMSCs, is consistent with the possibility that increased ColXV expression in response to hypoxia is mediated by an early network that supports the osteogenic potential of the cells. CONCLUSION: These results add useful information to understand the role of a still little investigated collagen such as ColXV, and identify ColXV as a marker of successful hypoxia preconditioning. As a whole, our data give further evidence that hypoxia preconditioned hMSCs have greater osteopotency than normal hMSCs, and that the effects of hypoxic regulation of hMSCs activities should be considered before they are clinically applied.

Immunoelectron microscopic localization of Collagen type XV during human mesenchymal stem cells mineralization.

Purpose/Aim of the study. Collagen type XV (ColXV) was identified, in our previews studies, as a novel component of bone extracellular matrix. The present study aims to investigate ColXV localization during mineralization of osteodifferentiated human mesenchymal stem cells (hMSCs). MATERIAL AND METHODS: hMSCs cultured in osteogenic medium have been analyzed at day 14 and 28 for mineral matrix deposition by alizarin red S staining, ultrastructural analysis and ColXV localization by immunogold electron microscopy. RESULTS: Our data show an intimate association between ColXV and fibrillar components in areas localized far from mineralized nodules. CONCLUSIONS: We have demonstrated the efficacy of ultrastructural analysis, combined with immunocytochemistry, to establish a temporal and spatial localization of ColXV. This data, added to previous evidences, contribute to validate the negative effects of calcium deposits on ColXV expression.

Emerging potential of gene silencing approaches targeting anti-chondrogenic factors for cell-based cartilage repair.

The field of cartilage repair has exponentially been growing over the past decade. Here, we discuss the possibility to achieve satisfactory regeneration of articular cartilage by means of human mesenchymal stem cells (hMSCs) depleted of anti-chondrogenic factors and implanted in the site of injury. Different types of molecules including transcription factors, transcriptional co-regulators, secreted proteins, and microRNAs have recently been identified as negative modulators of chondroprogenitor differentiation and chondrocyte function. We review the current knowledge about these molecules as potential targets for gene knockdown strategies using RNA interference (RNAi) tools that allow the specific suppression of gene function. The critical issues regarding the optimization of the gene silencing approach as well as the delivery strategies are discussed. We anticipate that further development of these techniques will lead to the generation of implantable hMSCs with enhanced potential to regenerate articular cartilage damaged by injury, disease, or aging.

Collagen type XV and the 'osteogenic status'.

We have previously demonstrated that collagen type XV (ColXV) is a novel bone extracellular matrix (ECM) protein. It is well known that the complex mixture of multiple components present in ECM can help both to maintain stemness or to promote differentiation of stromal cells following change in qualitative characteristics or concentrations. We investigated the possible correlation between ColXV expression and mineral matrix deposition by human mesenchymal stromal cells (hMSCs) with different osteogenic potential and by osteoblasts (hOBs) that are able to grow in culture medium with or without calcium. Analysing the osteogenic process, we have shown that ColXV basal levels are lower in cells less prone to osteo-induction such as hMSCs from Wharton Jelly (hWJMSCs), compared to hMSCs that are prone to osteo-induction such as those from the bone marrow (hBMMSCs). In the group of samples identified as 'mineralized MSCs', during successful osteogenic induction, ColXV protein continued to be detected at substantial levels until early stage of differentiation, but it significantly decreased and then disappeared at the end of culture when the matrix formed was completely calcified. The possibility to grow hOBs in culture medium without calcium corroborated the results obtained with hMSCs demonstrating that calcium deposits organized in a calcified matrix, and not calcium 'per se', negatively affected ColXV expression. As a whole, our data suggest that ColXV may participate in ECM organization in the early-phases of the osteogenic process and that this is a prerequisite to promote the subsequent deposition of mineral matrix.

Allogeneic cell transplant expands bone marrow distribution by colonizing previously abandoned areas: an FDG PET/CT analysis.

Mechanisms of hematopoietic reconstitution after bone marrow (BM) transplantation remain largely unknown. We applied a computational quantification software application to hybrid 18F-fluorodeoxyglucose positron emission tomography (PET)/computed tomography (CT) images to assess activity and distribution of the hematopoietic system throughout the whole skeleton of recently transplanted patients. Thirty-four patients underwent PET/CT 30 days after either adult stem cell transplantation (allogeneic cell transplantation [ACT]; n = 18) or cord blood transplantation (CBT; n = 16). Our software automatically recognized compact bone volume and trabecular bone volume (IBV) in CT slices. Within IBV, coregistered PET data were extracted to identify the active BM (ABM) from the inactive tissue. Patients were compared with 34 matched controls chosen among a published normalcy database. Whole body ABM increased in ACT and CBT when compared with controls (12.4 ± 3 and 12.8 ± 6.8 vs 8.1 ± 2.6 mL/kg of ideal body weight [IBW], P < .001). In long bones, ABM increased three- and sixfold in CBT and ACT, respectively, compared with controls (0.9 ± 0.9 and 1.7 ± 2.5 vs 0.3 ± 0.3 mL/kg IBW, P < .01). These data document an unexpected distribution of transplanted BM into previously abandoned BM sites.

Silencing of Antichondrogenic MicroRNA-221 in Human Mesenchymal Stem Cells Promotes Cartilage Repair In Vivo.

There is a growing demand for the development of experimental strategies for efficient articular cartilage repair. Current tissue engineering-based regenerative strategies make use of human mesenchymal stromal cells (hMSCs). However, when implanted in a cartilage defect, control of hMSCs differentiation toward the chondrogenic lineage remains a significant challenge. We have recently demonstrated that silencing the antichondrogenic regulator microRNA-221 (miR-221) was highly effective in promoting in vitro chondrogenesis of monolayered hMSCs in the absence of the chondrogenic induction factor TGF-ß. Here we investigated the feasibility of this approach first in conventional 3D pellet culture and then in an in vivo model. In pellet cultures, we observed that miR-221 silencing was sufficient to drive hMSCs toward chondrogenic differentiation in the absence of TGF-ß. In vivo, the potential of miR-221 silenced hMSCs was investigated by first encapsulating the cells in alginate and then by filling a cartilage defect in an osteochondral biopsy. After implanting the biopsy subcutaneously in nude mice, we found that silencing of miR-221 strongly enhanced in vivo cartilage repair compared to the control conditions (untreated hMSCs or alginate-only). Notably, miR-221 silenced hMSCs generated in vivo a cartilaginous tissue with no sign of collagen type X deposition, a marker of undesired hypertrophic maturation. Altogether our data indicate that silencing miR-221 has a prochondrogenic role in vivo, opening new possibilities for the use of hMSCs in cartilage tissue engineering. Stem Cells 2016;34:1801-1811.

Early identification of MCI converting to AD: a FDG PET study.

PURPOSE: Mild cognitive impairment (MCI) is a transitional pathological stage between normal ageing (NA) and Alzheimer's disease (AD). Although subjects with MCI show a decline at different rates, some individuals remain stable or even show an improvement in their cognitive level after some years. We assessed the accuracy of FDG PET in discriminating MCI patients who converted to AD from those who did not. METHODS: FDG PET was performed in 42 NA subjects, 27 MCI patients who had not converted to AD at 5 years (nc-MCI; mean follow-up time 7.5 ± 1.5 years), and 95 MCI patients who converted to AD within 5 years (MCI-AD; mean conversion time 1.8 ± 1.1 years). Relative FDG uptake values in 26 meta-volumes of interest were submitted to ANCOVA and support vector machine analyses to evaluate regional differences and discrimination accuracy. RESULTS: The MCI-AD group showed significantly lower FDG uptake values in the temporoparietal cortex than the other two groups. FDG uptake values in the nc-MCI group were similar to those in the NA group. Support vector machine analysis discriminated nc-MCI from MCI-AD patients with an accuracy of 89% (AUC 0.91), correctly detecting 93% of the nc-MCI patients. CONCLUSION: In MCI patients not converting to AD within a minimum follow-up time of 5 years and MCI patients converting within 5 years, baseline FDG PET and volume-based analysis identified those who converted with an accuracy of 89%. However, further analysis is needed in patients with amnestic MCI who convert to a dementia other than AD.

Non-invasive measurement of coronary flow reserve: uniqueness of radionuclide methods and alternative techniques.

Over the last several decades, radionuclide Myocardial Perfusion Imaging (MPI) has been a mainstay for the evaluation of coronary artery disease (CAD), based on the assumption that a detailed knowledge of stenosis localization and severity is not sufficient for clinical decision making. Furthermore, radionuclide MPI diagnostic accuracy has been implemented by the assessment of Coronary Flow Reserve (CFR) and Myocardial Blood Flow (MBF), as quantitative indexes of stenosis severity and surrogates of total ischaemic burden. Several considerations indicate that these measurement actually improve description of coronary physiology with respect to conventional qualitative image analysis. However, several alternative approaches have been optimized and increasingly proposed to achieve this task in the clinical setting. The aim of the present narrative review is to discuss strengths and weaknesses of the various cardiac modalities proposed to define CFR and MBF in the era of multi-modality imaging.

hnRNP K in PU.1-containing complexes recruited at the CD11b promoter: a distinct role in modulating granulocytic and monocytic differentiation of AML-derived cells.

PU.1 is essential for the differentiation of haemopoietic precursors and is strongly implicated in leukaemogenesis, yet the protein interactions that regulate its activity in different myeloid lineages are still largely unknown. In the present study, by combining fluorescent EMSA (electrophoretic mobility-shift assay) with MS, we reveal the presence of hnRNP K (heterogeneous nuclear ribonucleoprotein K) in molecular complexes that PU.1 forms on the CD11b promoter during the agonist-induced maturation of AML (acute myeloid leukaemia)-derived cells along both the granulocytic and the monocytic lineages. Although hnRNP K and PU.1 act synergistically during granulocytic differentiation, hnRNP K seems to have a negative effect on PU.1 activity during monocytic maturation. Since hnRNP K acts as a docking platform, integrating signal transduction pathways to nucleic acid-directed processes, it may assist PU.1 in activating or repressing transcription by recruiting lineage-specific components of the transcription machinery. It is therefore possible that hnRNP K plays a key role in the mechanisms underlying the specific targeting of protein-protein interactions identified as mediators of transcriptional activation or repression and may be responsible for the block of haemopoietic differentiation.

Adult advanced chronic lymphocytic leukemia: computational analysis of whole-body CT documents a bone structure alteration.

PURPOSE: To assess the presence of alteration of bone structure and bone marrow metabolism in adult patients who were suspected of having advanced chronic lymphocytic leukemia (ACLL) by using a computational prognostic model that was based on computational analysis of positron emission tomography (PET)/computed tomography (CT) images. MATERIALS AND METHODS: In this retrospective study, all patients signed written informed consent as a requisite to undergo PET/CT examination. However, due to its observational nature, approval from the ethical committee was not deemed necessary. Twenty-two previously untreated chronic lymphocytic leukemia patients underwent PET/CT for disease progression. PET/CT images were analyzed by using dedicated software, capable of recognizing an external 2-pixel bone ring whose Hounsfield coefficient served as cutoff to recognize trabecular and compact bone. PET/CT data from 22 age- and sex-matched control subjects were used as comparison. All data are reported as means ± standard deviations. The Student t test, log-rank, or Cox proportional hazards model were used as appropriate, considering a difference with a P value of less than .05 as significant. RESULTS: Trabecular bone was expanded in ACLL patients and occupied a larger fraction of the skeleton with respect to control subjects (mean, 39% ± 5 [standard deviation] vs 31% ± 7; ie, 32 of 81 mL/kg of ideal body weight vs 27 of 86 mL/kg of ideal body weight, respectively; P < .001). After stratification according to median value, patients with a ratio of trabecular to skeletal bone volume of more than 37.3% showed an actuarial 2-year survival of 18%, compared with 82% for those with a ratio of less than 37.3% (P < .001), independent from age, sex, biological markers, and disease duration. CONCLUSION: These data suggest that computational assessment of skeletal alterations might represent a new window for prediction of the clinical course of the disease.

The NFATc1/P2X7 receptor relationship in human intervertebral disc cells.

A comprehensive understanding of the molecules that play key roles in the physiological and pathological homeostasis of the human intervertebral disc (IVD) remains challenging, as does the development of new therapeutic treatments. We recently found a positive correlation between IVD degeneration (IDD) and P2X7 receptor (P2X7R) expression increases both in the cytoplasm and in the nucleus. Using immunocytochemistry, reverse transcription PCR (RT-PCR), overexpression, and chromatin immunoprecipitation, we found that NFATc1 and hypoxia-inducible factor-1α (HIF-1α) are critical regulators of P2X7R. Both transcription factors are recruited at the promoter of the P2RX7 gene and involved in its positive and negative regulation, respectively. Furthermore, using the proximity ligation assay, we revealed that P2X7R and NFATc1 form a molecular complex and that P2X7R is closely associated with lamin A/C, a major component of the nuclear lamina. Collectively, our study identifies, for the first time, P2X7R and NFATc1 as markers of IVD degeneration and demonstrates that both NFATc1 and lamin A/C are interaction partners of P2X7R.

Wharton's jelly-derived multifunctional hydrogels: New tools to promote intervertebral disc regeneration in vitro and ex vivo.

The degeneration of intervertebral disc (IVD) is a disease of the entire joint between two vertebrae in the spine caused by loss of extracellular matrix (ECM) integrity, to date with no cure. The various regenerative approaches proposed so far have led to very limited successes. An emerging opportunity arises from the use of decellularized ECM as a scaffolding material that, directly or in combination with other materials, has greatly facilitated the advancement of tissue engineering. Here we focused on the decellularized matrix obtained from human umbilical cord Wharton's jelly (DWJ) which retains several structural and bioactive molecules very similar to those of the IVD ECM. However, being a viscous gel, DWJ has limited ability to retain ordered structural features when considered as architecture scaffold. To overcome this limitation, we produced DWJ-based multifunctional hydrogels, in the form of 3D millicylinders containing different percentages of alginate, a seaweed-derived polysaccharide, and gelatin, denatured collagen, which may impart mechanical integrity to the biologically active DWJ. The developed protocol, based on a freezing step, leads to the consolidation of the entire polymeric dispersion mixture, followed by an ionic gelation step and a freeze-drying process. Finally, a porous, stable, easily storable, and suitable matrix for ex vivo experiments was obtained. The properties of the millicylinders (Wharton's jelly millicylinders [WJMs]) were then tested in culture of degenerated IVD cells isolated from disc tissues of patients undergoing surgical discectomy. We found that WJMs with the highest percentage of DWJ were effective in supporting cell migration, restoration of the IVD phenotype (increased expression of Collagen type 2, aggrecan, Sox9 and FOXO3a), anti-inflammatory action, and stem cell activity of resident progenitor/notochordal cells (increased number of CD24 positive cells). We are confident that the DWJ-based formulations proposed here can provide adequate stimuli to the cells present in the degenerated IVD to restart the anabolic machinery.

Exploiting stem cell therapy: the 3rd meeting of stem cell research Italy.

The study of stem cells is one of the most exciting areas of contemporary biomedical research. During the 3rd Joint Meeting of Stem Cell Research Italy (June 2012, Ferrara, Italy), scientists from different multidisciplinary areas explored new frontiers of basic and applied stem cell research with key lectures and oral presentations. There was a public debate on ethics during the opening ceremony, specifically on the limits and potentialities of adult and embryonic stem cells. Some scientists presented basic research data showing evolutionary aspects, which could be of interest in understanding specific biological phenomena. Others focused on "dangerous liaisons" between gene transfer vectors and the human genome. Some speakers provided insight into current stem cell therapies, such as those involving human epithelial stem cells for treatment of skin diseases. Other researchers presented data on close-to-therapy findings, such as the use of mesenchymal stem cells in brain repair. Of note, during the meeting, spotlights were focused on major issues that have to be considered for GMP stem cell production for cell therapy. In "Meet the Expert" sessions, specialists presented innovative technologies such as a next-generation sequencing system. Finally, the meeting provided an excellent opportunity for young scientists to show their findings, and to discuss with each other and with internationally recognized experts.