Community intervention trial for estimating risk of acute gastrointestinal illness from groundwater-supplied non-disinfected drinking water.

By community intervention in 14 non-disinfecting municipal water systems, we quantified sporadic acute gastrointestinal illness (AGI) attributable to groundwater. Ultraviolet (UV) disinfection was installed on all supply wells of intervention communities. In control communities, residents continued to drink non-disinfected groundwater. Intervention and control communities switched treatments by moving UV disinfection units at the study midpoint (crossover design). Study participants (n = 1,659) completed weekly health diaries during four 12-week surveillance periods. Water supply wells were analyzed monthly for enteric pathogenic viruses. Using the crossover design, groundwater-borne AGI was not observed. However, virus types and quantity in supply wells changed through the study, suggesting that exposure was not constant. Alternatively, we compared AGI incidence between intervention and control communities within the same surveillance period. During Period 1, norovirus contaminated wells and AGI attributable risk from well water was 19% (95% CI, -4%, 36%) for children <5 years and 15% (95% CI, -9%, 33%) for adults. During Period 3, echovirus 11 contaminated wells and UV disinfection slightly reduced AGI in adults. Estimates of AGI attributable risks from drinking non-disinfected groundwater were highly variable, but appeared greatest during times when supply wells were contaminated with specific AGI-etiologic viruses.

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.

Changing Behavior, Attitudes, and Beliefs About Food Safety: A Scoping Review of Interventions Across the World and Implications for Empowering Consumers.

Foodborne illnesses are a global public health issue. Responsibility to prevent foodborne disease is shared by many actors along the food supply chain, including consumers. However, consumers often lack knowledge about food safety and behaviors that can reduce risk. Consumers are often targeted for interventions to address these gaps, but a current comprehensive analysis of such interventions globally by type, geography, and outcome is lacking in the literature. In addition, there is a need to understand how individual interventions could be broadened to include the relationships between consumers and other actors in the food system, and how targeted communication strategies can affect behavior. We conducted a rigorous scoping review to assess consumer-facing food safety interventions carried out globally over the past 20 years, and categorized and analyzed them by type of intervention, methods, and outcomes to understand which interventions might be effective in changing consumer behavior, knowledge, attitudes, beliefs, and perceptions on food safety. Ninety-two interventions were reviewed, the majority of which were published in the last 10 years in North America. Most target adults, and 25% are directed at women and mothers. Health or risk communication interventions are becoming increasingly common to move beyond skill-based education and address risk perceptions of food safety that might motivate consumers. Only two studies addressed risk perception in consumers to potentially change food handlers' behavior outside of the home. This review suggests that focusing on risk perception combined with strategies that leverage emotion and trusted sources, such as respected peers or family members, might be useful strategies for interventions.

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.

The Public Health Impact of Implementing a Concentration-Based Microbiological Criterion for Controlling Salmonella in Ground Turkey.

Despite initiatives to improve the safety of poultry products in the United States, progress has stalled, and salmonellosis incidence is still above Healthy People 2020's goal. One strategy to manage Salmonella and verify process control in poultry establishments is to implement microbiological criteria (MC) linked to public health outcomes. Concentration-based MC have been used by the food industry; however, the public health impact of such approaches is only starting to be assessed. This study evaluated the public health impact of a concentration-based MC for Salmonella in raw ground turkey consumed in the United States using a quantitative risk assessment modeling approach. The distribution of Salmonella concentration in ground turkey was derived from USDA-FSIS monitoring surveys. Other variables and parameters were derived from public databases, literature, and expert opinion. Based on considered concentrations, implementing a MC of 1 cell/g led to an estimated 46.1% reduction (preventable fraction, PF) in the mean probability of illness when consumer cooking and cross-contamination were included. The PF was consistent across scenarios including or excluding cross-contamination and cooking, with slightly lower mean PF when cross-contamination was included. The proportion of lots not compliant with the 1 cell/g MC was 1.05% in the main scenarios and increased nonlinearly when higher Salmonella concentrations were assumed. Assumptions on concentration variability across lots and within lots had a large impact, highlighting the benefit of reducing this uncertainty. These approach and results can help inform the development of MC to monitor and control Salmonella in ground turkey products.

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.

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.

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.

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.

Modeling the risk of salmonellosis from consumption of pistachios produced and consumed in the United States.

The risk of salmonellosis from consumption of pistachios produced and consumed in the U.S. was assessed through quantitative microbial risk assessment. Data on Salmonella prevalence and concentration on pistachios, nut crop volume, storage times and temperatures during processing and handling, and reductions during storage or from roasting were derived from laboratory experiments, published literature, and industry expert opinion. Uncertainty was analyzed via what-if scenarios for Salmonella prevalence, concentration, storage reduction, treatment variability, portion of crop treated, and increased consumption. The estimated U.S. incidence of salmonellosis when 100% of pistachios were exposed to a 4 ± 0 log reduction treatment averaged 1.4 cases per billion servings, or <1 case/year, without considering Salmonella decline during storage. Including Salmonella decline during storage reduced the salmonellosis estimates approximately 10-fold. The predicted arithmetic mean number of cases associated with individual 500,000-kg storage silos, contaminated at the highest observed levels, ranged from 5 to 530 when the product was consumed untreated, but was reduced to below 1 case per silo when a 4 ± 0 log reduction treatment was applied. Assuming a uniform 4-log reduction treatment is applied to 100% of the crop and there is no decline of Salmonella during storage, the assessment indicates the following: 10-fold increases in either Salmonella prevalence or concentration, 2-fold increases in both prevalence and concentration, or consumption of >0.05% of untreated product volume yield an arithmetic mean risk of >1 case/year.

Transcriptional changes in innate immunity genes in head kidneys from Aeromonas salmonicida-challenged rainbow trout fed a mixture of polycyclic aromatic hydrocarbons.

We previously observed that exposure to a complex mixture of high molecular weight polycyclic aromatic hydrocarbons (PAHs) increased sensitivity of rainbow trout (Oncorhynchus mykiss) to subsequent challenge with Aeromonas salmonicida, the causative agent of furunculosis. In this study, we evaluate potential mechanisms associated with disease susceptibility from combined environmental factors of dietary PAH exposure and pathogen challenge. Rainbow trout were fed a mixture of ten high molecular weight PAHs at an environmentally relevant concentration (7.82µg PAH mixture/g fish/day) or control diet for 50 days. After 50 days of PAH exposure, fish were challenged with either Aeromonas salmonicida at a lethal concentration 30 (LC30) or growth media without the pathogen (mock challenge). Head kidneys were collected 2, 4, 10 and 20 days after challenge and gene expression (q<0.05) was evaluated among treatments. In animals fed the PAH contaminated diet, we observed down-regulation of expression for innate immune system genes in pathways (p<0.05) for the terminal steps of the complement cascade (complement component C6) and other bacteriolytic processes (lysozyme type II) potentially underlying increased disease susceptibility after pathogen challenge. Increased expression of genes associated with hemorrhage/tissue remodeling/inflammation pathways (p<0.05) was likely related to more severe head kidney damage due to infection in PAH-fed compared to control-fed fish. This study is the first to evaluate transcriptional signatures associated with the impact of chronic exposure to an environmentally relevant mixture of PAHs in disease susceptibility and immunity.

Transmission of Bacterial Zoonotic Pathogens between Pets and Humans: The Role of Pet Food.

Recent Salmonella outbreaks associated with dry pet food and treats raised the level of concern for these products as vehicle of pathogen exposure for both pets and their owners. The need to characterize the microbiological and risk profiles of this class of products is currently not supported by sufficient specific data. This systematic review summarizes existing data on the main variables needed to support an ingredients-to-consumer quantitative risk model to (1) describe the microbial ecology of bacterial pathogens in the dry pet food production chain, (2) estimate pet exposure to pathogens through dry food consumption, and (3) assess human exposure and illness incidence due to contact with pet food and pets in the household. Risk models populated with the data here summarized will provide a tool to quantitatively address the emerging public health concerns associated with pet food and the effectiveness of mitigation measures. Results of such models can provide a basis for improvements in production processes, risk communication to consumers, and regulatory action.

Modeling the long-term kinetics of Salmonella survival on dry pet food.

Due to multiple outbreaks and large-scale product recalls, Salmonella has emerged as a priority pathogen in dry pet food and treats. However, little data are available to quantify risks posed by these classes of products to both pets and their owners. Specifically, the kinetics of Salmonella survival on complex pet food matrices are not available. This study measured the long-term kinetics of Salmonella survival on a dry pet food under storage conditions commonly encountered during production, retail, and in households (aw < 0.60, 23 °C). A Salmonella enterica cocktail of 12 strains isolated from dry pet foods and treats was used to inoculate commercial dry dog food. Salmonella was enumerated on non-selective (BHI) and selective (XLD and BS) media. Results at 570 days indicated an initial relatively rapid decline (up to 54 days), followed by a much slower extended decline phase. The Weibull model provided a satisfactory fit for time series of Log-transformed Salmonella counts from all three media (δ: mean 4.65 day/Log (CFU/g); p: mean 0.364 on BHI). This study provides a survival model that can be applied in quantitative risk assessment models.

Dynamics of Escherichia coli Virulence Factors in Dairy Herds and Farm Environments in a Longitudinal Study in the United States.

Pathogenic Escherichia coli or its associated virulence factors have been frequently detected in dairy cow manure, milk, and dairy farm environments. However, it is unclear what the long-term dynamics of E. coli virulence factors are and which farm compartments act as reservoirs. This study assessed the occurrence and dynamics of four E. coli virulence factors (eae, stx1, stx2, and the gamma allele of the tir gene [γ-tir]) on three U.S. dairy farms. Fecal, manure, water, feed, milk, and milk filter samples were collected from 2004 to 2012. Virulence factors were measured by postenrichment quantitative PCR (qPCR). All factors were detected in most compartments on all farms. Fecal and manure samples showed the highest prevalence, up to 53% for stx and 21% for γ-tir in fecal samples and up to 84% for stx and 44% for γ-tir in manure. Prevalence was low in milk (up to 1.9% for stx and 0.7% for γ-tir). However, 35% of milk filters were positive for stx and 20% were positive for γ-tir. All factors were detected in feed and water. Factor prevalence and levels, expressed as qPCR cycle threshold categories, fluctuated significantly over time, with no clear seasonal signal independent from year-to-year variability. Levels were correlated between fecal and manure samples, and in some cases autocorrelated, but not between manure and milk filters. Shiga toxins were nearly ubiquitous, and 10 to 18% of the lactating cows were potential shedders of E. coli O157 at least once during their time in the herds. E. coli virulence factors appear to persist in many areas of the farms and therefore contribute to transmission dynamics.

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.

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.

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.