Unorthodox localization of P2X7 receptor in subcellular compartments of skeletal system cells.

Identifying the subcellular localization of a protein within a cell is often an essential step in understanding its function. The main objective of this report was to determine the presence of the P2X7 receptor (P2X7R) in healthy human cells of skeletal system, specifically osteoblasts (OBs), chondrocytes (Chs) and intervertebral disc (IVD) cells. This receptor is a member of the ATP-gated ion channel family, known to be a main sensor of extracellular ATP, the prototype of the danger signal released at sites of tissue damage, and a ubiquitous player in inflammation and cancer, including bone and cartilaginous tissues. Despite overwhelming data supporting a role in immune cell responses and tumor growth and progression, a complete picture of the pathophysiological functions of P2X7R, especially when expressed by non-immune cells, is lacking. Here we show that human wild-type P2X7R (P2X7A) was expressed in different samples of human osteoblasts, chondrocytes and intervertebral disc cells. By fluorescence microscopy (LM) and immunogold transmission electron microscopy we localized P2X7R not only in the canonical sites (plasma membrane and cytoplasm), but also in the nucleus of all the 3 cell types, especially IVD cells and OBs. P2X7R mitochondrial immunoreactivity was predominantly detected in OBs and IVD cells, but not in Chs. Evidence of subcellular localization of P2X7R may help to i. understand the participation of P2X7R in as yet unidentified signaling pathways in the joint and bone microenvironment, ii. identify pathologies associated with P2X7R mislocalization and iii. design specific targeted therapies.

Pro­differentiating compounds for human intervertebral disc cells are present in Violina pumpkin leaf extracts.

Intervertebral disc (IVD) degeneration (IDD) is closely associated with inflammation, oxidative stress and loss of the discogenic phenotype, which current therapies are unable to reverse. In the present study, the effects of acetone extract from Violina pumpkin (Cucurbita moschata) leaves on degenerated IVD cells were investigated. IVD cells were isolated from the degenerated disc tissue of patients undergoing spinal surgery and were exposed to acetone extract and three major thin layer chromatography subfractions. The results revealed that, in particular, the cells benefited from exposure to subfraction Fr7, which consisted almost entirely of p­Coumaric acid. Western blot and immunocytochemical analysis showed that Fr7 induced a significant increase in discogenic transcription factors (SOX9 and tricho­rhino­phalangeal syndrome type I protein, zinc finger protein), extracellular matrix components (aggrecan, collagen type II), cellular homeostasis and stress response regulators, such as FOXO3a, nuclear factor erythroid 2­related factor 2, superoxide dismutase 2 and sirtuin 1. Two important markers related to the presence and activity of stem cells, migratory capacity and OCT4 expression, were assessed by scratch assay and western blotting, respectively, and were significantly increased in Fr7­treated cells. Moreover, Fr7 counteracted H2O2­triggered cell damage, preventing increases in the pro­inflammatory and anti­chondrogenic microRNA (miR), miR­221. These findings strengthen the hypothesis that adequate stimuli can support resident cells to repopulate the degenerated IVD and restart the anabolic machinery. Taken together, these data contribute to the discovery of molecules potentially effective in slowing the progression of IDD, a disease for which there is currently no effective treatment. Moreover, the use of part of a plant, the pumpkin leaves, which is usually considered a waste product in the Western world, indicated that it contains substances with potential beneficial effects on human health.

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.

General consensus on multimodal functions and validation analysis of perinatal derivatives for regenerative medicine applications.

Perinatal tissues, such as placenta and umbilical cord contain a variety of somatic stem cell types, spanning from the largely used hematopoietic stem and progenitor cells to the most recently described broadly multipotent epithelial and stromal cells. As perinatal derivatives (PnD), several of these cell types and related products provide an interesting regenerative potential for a variety of diseases. Within COST SPRINT Action, we continue our review series, revising and summarizing the modalities of action and proposed medical approaches using PnD products: cells, secretome, extracellular vesicles, and decellularized tissues. Focusing on the brain, bone, skeletal muscle, heart, intestinal, liver, and lung pathologies, we discuss the importance of potency testing in validating PnD therapeutics, and critically evaluate the concept of PnD application in the field of tissue regeneration. Hereby we aim to shed light on the actual therapeutic properties of PnD, with an open eye for future clinical application. This review is part of a quadrinomial series on functional/potency assays for validation of PnD, spanning biological functions, such as immunomodulation, anti-microbial/anti-cancer, anti-inflammation, wound healing, angiogenesis, and regeneration.

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.

Comparison Between 18F-FDG PET-Based and CT-Based Criteria in Non-Small Cell Lung Cancer Patients Treated with Nivolumab.

Because of the peculiar mechanism of action of immune checkpoint inhibitors (ICIs), evaluation of the radiologic response to them in solid tumors presents many challenges. We aimed to compare evaluation of the first response to nivolumab by means of CT-based criteria with respect to 18F-FDG PET response criteria in non-small cell lung cancer (NSCLC) patients. Methods: Seventy-two patients with advanced NSCLC were recruited in a single-institution ancillary trial within the expanded-access program (NCT02475382) for nivolumab. Patients underwent CT and 18F-FDG PET at baseline and after 4 cycles (the first evaluation). In cases of progressive disease, an additional evaluation was performed after 2 further cycles to confirm progression. We evaluated the treatment response on CT using RECIST 1.1 and the immune-related response criteria (irRC) and on 18F-FDG PET using PERCIST and immunotherapy-modified PERCIST. The concordance between CT- and PET-based criteria and the capability of each method to predict overall survival were evaluated. Results: Forty-eight of 72 patients were evaluable for a first response assessment with both PET- and CT-based criteria. We observed low concordance between CT- and PET-based criteria (κ-value of 0.346 and 0.355 between PERCIST and imPERCIST and RECIST, respectively. κ-value of 0.128 and 0.198 between PERCIST and imPERCIST and irRC, respectively). Regarding overall survival, irRC could more reliably distinguish responders from nonresponders. However, thanks to the prognostic value of partial metabolic response assessed by both PERCIST and immunotherapy-modified PERCIST, PET-based response maintained prognostic significance in patients classified as having progressive disease on the basis of irRC. Conclusion: Even though the present study did not support the routine use of 18F-FDG PET in the general population of NSCLC patients treated with ICIs, the findings suggest that metabolic response assessment has added prognostic value, potentially improving therapeutic decision making.

SLUG/HIF1-α/miR-221 regulatory circuit in endometrial cancer.

BACKGROUND AND PURPOSE: The pathogenesis of endometrial cancer (EC) involves many regulatory pathways including transcriptional regulatory networks supported by transcription factors and microRNAs only in part known. The aim of this retrospective study was to explore the possible correlation in the EC microenvironment between master regulators of complex phenomena such as steroid responsiveness through estrogen receptor alpha (ERα) and progesterone receptor (PR), epithelial-to-mesenchymal transition (supported by SLUG transcription factor), hypoxia (with hypoxia inducible factor-1 alpha, HIF-1α), and obesity that has been recognized as a EC risk factor. METHODS: Formalin-Fixed Paraffin-Embedded (FFPE) blocks from University of Ferrara Pathology Archive were used and allocated into 2 groups according to their immunohistochemical positivity to ERα and PR, distinguishing the samples with a more benign prognosis (ERα+/PR+) from those with a poorer prognosis (ERα-/PR-). Immunohistochemistry for HIF1-α and SLUG was also performed. Body mass index (BMI) was registered at the time of diagnosis: patients with BMI ≥ 30 kg/m2 were defined obese (OB). Total RNA was isolated for miR-221 analysis. RESULTS: We showed a comparable percentage of HIF1-α and SLUG positive samples in the ERα+/PR+ and ERα-/PR- groups. However, the obesity factor impacted more in the ERα+/PR+ group since the ratio between OB and non-obese (NOB) patients with high expression of HIF1-α and SLUG was higher in ERα+/PR+ than in the ERα-/PR- group. miR-221 levels were significantly higher in the OB than NOB patients, and, also in this case, obesity impacted more in the ERα+/PR+ group. CONCLUSIONS: A molecular circuit of mutual regulation between ERα, PR, HIF1-α, SLUG and miR-221 is feasible in the EC and was firstly suggested by our research. In this interplay miR-221 seems to be in a nodal point of the regulatory system that is particularly strengthened by the metabolic changes in obesity.

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.

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.

Upregulation of the alternative splicing factor NOVA2 in colorectal cancer vasculature.

BACKGROUND: Tumor-specific isoforms generated by alternative splicing (AS) are demonstrated to contribute to tumor progression and can represent potential biomarkers. NOVA2 is an AS factor that in physiological conditions regulates endothelial cells' (ECs) polarity and vessel lumen maturation, likely by mediating AS of apical-basal polarity regulators. However, NOVA2 expression in tumor ECs and its regulation have never been investigated. METHODS: To elucidate this, 40 colorectal cancer patients were enrolled and NOVA2 expression was investigated by immunohistochemistry in samples bearing both the normal mucosa and the tumor tissue. RESULTS: NOVA2 was found expressed in ECs of tumor vasculature and, importantly, it was upregulated in tumor ECs with respect to normal mucosa ECs in all cases (P<0.001). The same samples analyzed by immunohistochemistry for the expression HIF1α, a marker of hypoxia, showed a positive and significant association with NOVA2 levels (P=0.045). Of note, NOVA2 was upregulated by hypoxia also in an in vitro ECs model. CONCLUSION: Our results provide, for the first time, evidence of NOVA2 expression and upregulation in tumor ECs and highlight hypoxia as a potential regulatory factor. These findings open a completely new perspective to study tumor vasculature and to uncover NOVA2 as a potential source of biomarkers and therapeutic targets based on AS isoforms.

Assessment of Skeletal Tumor Load in Metastasized Castration-Resistant Prostate Cancer Patients: A Review of Available Methods and an Overview on Future Perspectives.

Metastasized castration-resistant prostate cancer (mCRPC), is the most advanced form of prostate neoplasia, where massive spread to the skeletal tissue is frequent. Patients with this condition are benefiting from an increasing number of treatment options. However, assessing tumor response in patients with multiple localizations might be challenging. For this reason, many computational approaches have been developed in the last decades to quantify the skeletal tumor burden and treatment response. In this review, we analyzed the progressive development and diffusion of such approaches. A computerized literature search of the PubMed/Medline was conducted, including articles between January 2008 and March 2018. The search was expanded by manually reviewing the reference list of the chosen articles. Thirty-five studies were identified. The number of eligible studies greatly increased over time. Studies could be categorized in the following categories: automated analysis of 2D scans, SUV-based thresholding, hybrid CT- and SUV-based thresholding, and MRI-based thresholding. All methods are discussed in detail. Automated analysis of bone tumor burden in mCRPC is a growing field of research; when choosing the appropriate method of analysis, it is important to consider the possible advantages as well as the limitations thoroughly.

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.

Metabolic correlates of reserve and resilience in MCI due to Alzheimer's Disease (AD).

BACKGROUND: We explored the presence of both reserve and resilience in late-converter mild cognitive impairment due to Alzheimer's disease (MCI-AD) and in patients with slowly progressing amyloid-positive MCI by assessing the topography and extent of neurodegeneration with respect to both "aggressive" and typically progressing phenotypes and in the whole group of patients with MCI, grounding the stratification on education level. METHODS: We analyzed 94 patients with MCI-AD followed until conversion to dementia and 39 patients with MCI who had brain amyloidosis (AMY+ MCI), all with available baseline 18F-fluorodeoxyglucose positron emission tomography (FDG-PET) results. Using a data-driven approach based on conversion time, patients with MCI-AD were divided into typical AD and late-converter subgroups. Similarly, on the basis of annual rate of Mini Mental State Examination score reduction, AMY+ MCI group was divided, obtaining smoldering (first tertile) and aggressive (third tertile) subgroups. Finally, we divided the whole group (MCI-AD and AMY+ MCI) according to years of schooling, obtaining four subgroups: poorly educated (Low-EDUC; first quartile), patients with average education (Average-EDUC; second quartile), highly educated (High-EDUC; third quartile), and exceptionally educated (Except-EDUC; fourth quartile). FDG-PET of typical AD, late converters, and aggressive and smoldering AMY+ MCI subgroups, as well as education level-based subgroups, were compared with healthy volunteer control subjects (CTR) and within each group using a two-samples t test design (SPM8; p < 0.05 family-wise error-corrected). RESULTS: Late converters were characterized by relatively preserved metabolism in the right middle temporal gyrus (Brodmann area [BA] 21) and in the left orbitofrontal cortex (BA 47) with respect to typical AD. When compared with CTR, the High-EDUC subgroup demonstrated a more extended bilateral hypometabolism in the posterior parietal cortex, posterior cingulate cortex, and precuneus than the Low- and Average-EDUC subgroups expressing the same level of cognitive impairment. The Except-EDUC subgroup showed a cluster of significant hypometabolism including only the left posterior parietal cortex (larger than the Low- and Average-EDUC subgroups but not further extended with respect to the High-EDUC subgroup). CONCLUSIONS: Middle and inferior temporal gyri may represent sites of resilience rather than a hallmark of a more aggressive pattern (when hypometabolic). These findings thus support the existence of a relatively homogeneous AD progression pattern of hypometabolism despite AD heterogeneity and interference of cognitive reserve. In fact, cortical regions whose "metabolic resistance" was associated with slower clinical progression had different localization with respect to the regions affected by education-related reserve.

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.

Menaquinone-4 enhances osteogenic potential of human amniotic fluid mesenchymal stem cells cultured in 2D and 3D dynamic culture systems.

Menaquinones, also known as Vitamin K2 family, regulate calcium homeostasis in a 'bone-vascular cross-talk' and recently received particular attention for their positive effect on bone formation. Given that the correlation between menaquinones and bone metabolism to date is still unclear, the objective of our study was to investigate the possible role of menaquinone-4 (MK-4), an isoform of the menaquinones family, in the modulation of osteogenesis. For this reason, we used a model of human amniotic fluid mesenchymal stem cells (hAFMSCs) cultured both in two-dimensional (2D) and three-dimensional (3D; RCCS™bioreactor) in vitro culture systems. Furthermore, to mimic the 'bone remodelling unit' in vitro, hAFMSCs were co-cultured in the 3D system with human monocyte cells (hMCs) as osteoclast precursors. The results showed that in a conventional 2D culture system, hAFMSCs were responsive to the MK-4, which significantly improved the osteogenic process through γ-glutamyl carboxylase-dependent pathway. The same results were obtained in the 3D dynamic system where MK-4 treatment supported the osteoblast-like formation promoting the extracellular bone matrix deposition and the expression of the osteogenic-related proteins (alkaline phosphatase, osteopontin, collagen type-1 and osteocalcin). Notably, when the hAFMSCs were co-cultured in a 3D dynamic system with the hMCs, the presence of MK-4 supported the cellular aggregate formation as well as the osteogenic function of hAFMSCs, but negatively affected the osteoclastogenic process. Taken together, our results demonstrate that MK-4 supported the aggregate formation of hAFMSCs and increased the osteogenic functions. Specifically, our data could help to optimize bone regenerative medicine combining cell-based approaches with MK-4 treatment.

The expression of cystathionine gamma-lyase is regulated by estrogen receptor alpha in human osteoblasts.

Hydrogen sulfide (H2S), generated in the osteoblasts predominantly via cystathionine-γ-lyase (CSE), is bone protective. Previous studies suggested that the onset of bone loss due to estrogen deficiency is associated to decreased levels of H2S and blunted gene expression of CSE. However, there are still a lot of unknowns on how H2S levels influence bone cells function. The present study aims to explore the mechanisms by which estrogen may regulate CSE expression, in particular the role of estrogen receptor alpha (ERα) in human osteoblasts (hOBs). Vertebral lamina derived hOBs were characterized and then assessed for CSE expression by western blot analysis in the presence or absence of ERα overexpression. Bioinformatic analysis, luciferase reporter assay and ChIP assay were performed to investigate ERα recruitment and activity on hCSE gene promoter. Three putative half Estrogen Responsive Elements (EREs) were identified in the hCSE core promoter and were found to participate in the ERα - mediated positive regulation of CSE expression. All osteoblast samples responded to ERα over-expression increasing the levels of CSE protein in a comparable manner. Notably, the ERα recruitment on the regulatory regions of the CSE promoter occurred predominantly in female hOBs than in male hOBs. The obtained results suggest that CSE/H2S system is in relation with estrogen signaling in bone in a gender specific manner.

Functional Activation of Osteoclast Commitment in Chronic Lymphocytic Leukaemia: a Possible Role for RANK/RANKL Pathway.

Skeletal erosion has been found to represent an independent prognostic indicator in patients with advanced stages of chronic lymphocytic leukaemia (CLL). Whether this phenomenon also occurs in early CLL phases and its underlying mechanisms have yet to be fully elucidated. In this study, we prospectively enrolled 36 consecutive treatment-naïve patients to analyse skeletal structure and bone marrow distribution using a computational approach to PET/CT images. This evaluation was combined with the analysis of RANK/RANKL loop activation in the leukemic clone, given recent reports on its role in CLL progression. Bone erosion was particularly evident in long bone shafts, progressively increased from Binet stage A to Binet stage C, and was correlated with both local expansion of metabolically active bone marrow documented by FDG uptake and with the number of RANKL + cells present in the circulating blood. In immune-deficient NOD/Shi-scid, γcnull (NSG) mice, administration of CLL cells caused an appreciable compact bone erosion that was prevented by Denosumab. CLL cell proliferation in vitro correlated with RANK expression and was impaired by Denosumab-mediated disruption of the RANK/RANKL loop. This study suggests an interaction between CLL cells and stromal elements able to simultaneously impair bone structure and increase proliferating potential of leukemic clone.

A Score-Based Approach to 18F-FDG PET Images as a Tool to Describe Metabolic Predictors of Myocardial Doxorubicin Susceptibility.

PURPOSE: To verify the capability of 18F-fluorodeoxy-glucose positron emission tomography/computed tomography (FDG-PET/CT) to identify patients at higher risk of developing doxorubicin (DXR)-induced cardiotoxicity, using a score-based image approach. METHODS: 36 patients underwent FDG-PET/CT. These patients had shown full remission after DXR-based chemotherapy for Hodgkin's disease (DXR dose: 40-50 mg/m² per cycle), and were retrospectively enrolled. Inclusion criteria implied the presence of both pre- and post-chemotherapy clinical evaluation encompassing electrocardiogram (ECG) and echocardiography. Myocardial metabolism at pre-therapy PET was evaluated according to both standardized uptake value (SUV)- and score-based approaches. The capability of the score-based image assessment to predict the occurrence of cardiac toxicity with respect to SUV measurement was then evaluated. RESULTS: In contrast to the SUV-based approach, the five-point scale method does not linearly stratify the risk of the subsequent development of cardiotoxicity. However, converting the five-points scale to a dichotomic evaluation (low vs. high myocardial metabolism), FDG-PET/CT showed high diagnostic accuracy in the prediction of cardiac toxicity (specificity = 100% and sensitivity = 83.3%). In patients showing high myocardial uptake at baseline, in which the score-based method is not able to definitively exclude the occurrence of cardiac toxicity, myocardial SUV mean quantification is able to further stratify the risk between low and intermediate risk classes. CONCLUSIONS: the score-based approach to FDG-PET/CT images is a feasible method for predicting DXR-induced cardiotoxicity. This method might improve the inter-reader and inter-scanner variability, thus allowing the evaluation of FDG-PET/CT images in a multicentral setting.

Comparative diagnostic accuracy of 18F-FDG PET/CT for breast cancer recurrence.

In the last decades, in addition to conventional imaging techniques and magnetic resonance imaging (MRI), 18F-fluorodeoxyglucose positron emission tomography/computed tomography (FDG PET/CT) has been shown to be relevant in the detection and management of breast cancer recurrence in doubtful cases in selected groups of patients. While there are no conclusive data indicating that imaging tests, including FDG PET/CT, produce a survival benefit in asymptomatic patients, FDG PET/CT can be useful for identifying the site of relapse when traditional imaging methods are equivocal or conflicting and for identifying or confirming isolated loco-regional relapse or isolated metastatic lesions. The present narrative review deals with the potential role of FDG PET in these clinical settings by comparing its accuracy and impact with conventional imaging modalities such as CT, ultrasound, bone scan, 18F-sodium fluoride PET/CT (18F-NaF PET/CT) as well as MRI. Patient-focused perspectives in terms of patients' satisfaction and acceptability are also discussed.

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.