A Computational Template for Three-Dimensional Modeling of the Vascular Scaffold of the Human Thyroid Gland

We recently designed an innovative scaffold-bioreactor unit for the bioengineering of a three-dimensional (3D) bioartificial human thyroid gland or its miniaturized replica as a part of a microfluidic chip test system.This device is based on the evidence that the 3D geometry of the intraglandular stromal/vascular scaffold (SVS; i.e., the fibrous and vascular matrix) of mammalian viscera plays a key role in guiding growth and differentiation of in vitro seeded cells. Therefore, we initiated a research program focused on computer-aided reconstruction of the 2nd to 4th order intralobar arterial network (IAN) of the human thyroid gland asa reliable surrogate for its 3D SVS, to be used as an input for rapid prototyping of a biomaterial replica. Tothis end, we developed a computational template that works within the Mathematica environment, giving rise to a quasi-fractal growth of the IAN distribution, constrained within an approximation of the thyroidl obe shape as a closed surface. Starting from edge detection of planar images of real human thyroid lobes acquired by in vivo real-time ultrasonography, we performed data approximation of the lobar profiles based on splines and Bezier curves, providing 3D lobar shapes as geometric boundaries for vessel growth by a diffusion-limited aggregation model. Our numerical procedures allowed for a robust connection between development of lobar arterial trees and thyroid lobe shape, led to a vascular self-similarity consistent with that of a cadaveric lobar arterial cast, and reproduced arterial vessels in a proportion not statistically different from that described for the real human thyroid gland. We conclude that our algorithmic template offers a reliable reproduction of the extremely complex IAN of the adult human thyroid lobe, potentially useful as a computational guidance for bioprinting of thyroid lobe matrix replicas. In addition, due to the simplicity and limited number of morphometrical parameters required by our system, we predict its application to the design of a number of patient-tailored human bioartificial organs and organs-on-chip,including parenchymal viscera and bones

Targeting T regulatory (Treg) cells in immunotherapy-resistant cancers

Primary or secondary (i.e., acquired) resistance is a common occurrence in cancer patients and is often associated with high numbers of T regulatory (Treg) cells (CD4+CD25+FOXP3+). The approval of ipilimumab and the development of similar pharmacological agents targeting cell surface proteins on Treg cells demonstrates that such intervention may overcome resistance in cancer patients. Hence, the clinical development and subsequent approval of Cytotoxic T Lymphocyte Antigen-4 (CTLA-4) targeting agents can serve as a prototype for similar agents. Such new agents aspire to be highly specific and have a reduced toxicity profile while increasing effector T cell function or effector T/T regulatory (Teff/Treg) ratio. While clinical development with large molecules has shown the greatest advancement, small molecule inhibitors that target immunomodulation are increasingly entering early clinical investigation. These new small molecule inhibitors often target specific intracellular signaling pathways [e.g., phosphoinositide-3-kinase delta (PI3K-δ)] that play an important role in regulating the function of Treg cells. This review will summarize the lessons currently applied to develop novel clinical agents that target Treg cells

A Smart Motor Rehabilitation System Based on the Internet of Things and Humanoid Robotics

The Internet of Things (IoT) is gaining increasing attention in healthcare due to its potential to enable continuous monitoring of patients, both at home and in controlled medical environments. In this paper, we explore the integration of IoT with human-robotics in the context of motor rehabilitation for groups of patients performing moderate physical routines, focused on balance, stretching, and posture. Specifically, we propose the I-TROPHYTS framework, which introduces a step-change in motor rehabilitation by advancing towards more sustainable medical services and personalized diagnostics. Our framework leverages wearable sensors to monitor patients’ vital signs and edge computing to detect and estimate motor routines. In addition, it incorporates a humanoid robot that mimics the actions of a physiotherapist, adapting motor routines in real-time based on the patient’s condition. All data from physiotherapy sessions are modeled using an ontology, enabling automatic reasoning and planning of robot actions. In this paper, we present the architecture of the proposed framework, which spans four layers, and discuss its enabling components. Furthermore, we detail the current deployment of the IoT system for patient monitoring and automatic identification of motor routines via Machine Learning techniques. Our experimental results, collected from a group of volunteers performing balance and stretching exercises, demonstrate that we can achieve nearly 100% accuracy in distinguishing between shoulder abduction and shoulder flexion, using Inertial Measurement Unit data from wearable IoT devices placed on the wrist and elbow of the test subjects

The mTOR and PP2A Pathways Regulate PHD2 Phosphorylation to Fine-Tune HIF1α Levels and Colorectal Cancer Cell Survival under Hypoxia

SummaryOxygen-dependent HIF1α hydroxylation and degradation are strictly controlled by PHD2. In hypoxia, HIF1α partly escapes degradation because of low oxygen availability. Here, we show that PHD2 is phosphorylated on serine 125 (S125) by the mechanistic target of rapamycin (mTOR) downstream kinase P70S6K and that this phosphorylation increases its ability to degrade HIF1α. mTOR blockade in hypoxia by REDD1 restrains P70S6K and unleashes PP2A phosphatase activity. Through its regulatory subunit B55α, PP2A directly dephosphorylates PHD2 on S125, resulting in a further reduction of PHD2 activity that ultimately boosts HIF1α accumulation. These events promote autophagy-mediated cell survival in colorectal cancer (CRC) cells. B55α knockdown blocks neoplastic growth of CRC cells in vitro and in vivo in a PHD2-dependent manner. In patients, CRC tissue expresses higher levels of REDD1, B55α, and HIF1α but has lower phospho-S125 PHD2 compared with a healthy colon. Our data disclose a mechanism of PHD2 regulation that involves the mTOR and PP2A pathways and controls tumor growth.Summary Oxygen-dependent HIF1α hydroxylation and degradation are strictly controlled by PHD2. In hypoxia, HIF1α partly escapes degradation because of low oxygen availability. Here, we show that PHD2 is phosphorylated on serine 125 (S125) by the mechanistic target of rapamycin (mTOR) downstream kinase P70S6K and that this phosphorylation increases its ability to degrade HIF1α. mTOR blockade in hypoxia by REDD1 restrains P70S6K and unleashes PP2A phosphatase activity. Through its regulatory subunit B55α, PP2A directly dephosphorylates PHD2 on S125, resulting in a further reduction of PHD2 activity that ultimately boosts HIF1α accumulation. These events promote autophagy-mediated cell survival in colorectal cancer (CRC) cells. B55α knockdown blocks neoplastic growth of CRC cells in vitro and in vivo in a PHD2-dependent manner. In patients, CRC tissue expresses higher levels of REDD1, B55α, and HIF1α but has lower phospho-S125 PHD2 compared with a healthy colon. Our data disclose a mechanism of PHD2 regulation that involves the mTOR and PP2A pathways and controls tumor growth

MET is required for the recruitment of anti-tumoural neutrophils

Mutations or amplification of the MET proto-oncogene are involved in the pathogenesis of several tumours, which rely on the constitutive engagement of this pathway for their growth and survival. However, MET is expressed not only by cancer cells but also by tumour-associated stromal cells, although its precise role in this compartment is not well characterized. Here we show that MET is required for neutrophil chemoattraction and cytotoxicity in response to its ligand hepatocyte growth factor (HGF). Met deletion in mouse neutrophils enhances tumour growth and metastasis. This phenotype correlates with reduced neutrophil infiltration to both the primary tumour and metastatic sites. Similarly, Met is necessary for neutrophil transudation during colitis, skin rash or peritonitis. Mechanistically, Met is induced by tumour-derived tumour necrosis factor (TNF)-α or other inflammatory stimuli in both mouse and human neutrophils. This induction is instrumental for neutrophil transmigration across an activated endothelium and for inducible nitric oxide synthase production upon HGF stimulation. Consequently, HGF/MET-dependent nitric oxide release by neutrophils promotes cancer cell killing, which abates tumour growth and metastasis. After systemic administration of a MET kinase inhibitor, we prove that the therapeutic benefit of MET targeting in cancer cells is partly countered by the pro-tumoural effect arising from MET blockade in neutrophils. Our work identifies an unprecedented role of MET in neutrophils, suggests a potential ‘Achilles’ heel’ of MET-targeted therapies in cancer, and supports the rationale for evaluating anti-MET drugs in certain inflammatory diseases

Woven bone formation and mineralization by rat mesenchymal stromal cells imply increased expression of the intermediate filament desmin

BackgroundDisordered and hypomineralized woven bone formation by dysfunctional mesenchymal stromal cells (MSCs) characterize delayed fracture healing and endocrine –metabolic bone disorders like fibrous dysplasia and Paget disease of bone. To shed light on molecular players in osteoblast differentiation, woven bone formation, and mineralization by MSCs we looked at the intermediate filament desmin (DES) during the skeletogenic commitment of rat bone marrow MSCs (rBMSCs), where its bone-related action remains elusive.ResultsMonolayer cultures of immunophenotypically- and morphologically - characterized, adult male rBMSCs showed co-localization of desmin (DES) with vimentin, F-actin, and runx2 in all cell morphotypes, each contributing to sparse and dense colonies. Proteomic analysis of these cells revealed a topologically-relevant interactome, focused on cytoskeletal and related enzymes//chaperone/signalling molecules linking DES to runx2 and alkaline phosphatase (ALP). Osteogenic differentiation led to mineralized woven bone nodules confined to dense colonies, significantly smaller and more circular with respect to controls. It significantly increased also colony-forming efficiency and the number of DES-immunoreactive dense colonies, and immunostaining of co-localized DES/runx-2 and DES/ALP. These data confirmed pre-osteoblastic and osteoblastic differentiation, woven bone formation, and mineralization, supporting DES as a player in the molecular pathway leading to the osteogenic fate of rBMSCs.ConclusionImmunocytochemical and morphometric studies coupled with proteomic and bioinformatic analysis support the concept that DES may act as an upstream signal for the skeletogenic commitment of rBMSCs. Thus, we suggest that altered metabolism of osteoblasts, woven bone, and mineralization by dysfunctional BMSCs might early be revealed by changes in DES expression//levels. Non-union fractures and endocrine – metabolic bone disorders like fibrous dysplasia and Paget disease of bone might take advantage of this molecular evidence for their early diagnosis and follow-up

Correlation between tooth decay and insulin resistance in normal weight males prompts a role for myo-inositol as a regenerative factor in dentistry and oral surgery: a feasibility study

BackgroundIn an era of precision and stratified medicine, homogeneity in population-based cohorts, stringent causative entry, and pattern analysis of datasets are key elements to investigate medical treatments. Adhering to these principles, we collected in vivo and in vitro data pointing to an insulin-sensitizing/insulin-mimetic effect of myo-inositol (MYO) relevant to cell regeneration in dentistry and oral surgery. Confirmation of this possibility was obtained by in silico analysis of the relation between in vivo and in vitro results (the so-called bed-to-benchside reverse translational approach).ResultsFourteen subjects over the 266 screened were young adult, normal weight, euglycemic, sedentary males having normal appetite, free diet, with a regular three-times-a-day eating schedule, standard dental hygiene, and negligible malocclusion/enamel defects. Occlusal caries were detected by fluorescence videoscanning, whereas body composition and energy balance were estimated with plicometry, predictive equations, and handgrip. Statistically significant correlations (Pearson r coefficient) were found between the number of occlusal caries and anthropometric indexes predicting insulin resistance (IR) in relation to the abdominal/visceral fat mass, fat-free mass, muscular strength, and energy expenditure adjusted to the fat and muscle stores. This indicated a role for IR in affecting dentin reparative processes. Consistently, in vitro administration of MYO to HUVEC and Swiss NIH3T3 cells in concentrations corresponding to those administered in vivo to reduce IR resulted in statistically significant cell replication (ANOVA/Turkey tests), suggesting that MYO has the potential to counteract inhibitory effects of IR on dental vascular and stromal cells turnover. Finally, in in silico experiments, quantitative evaluation (WOE and information value) of a bioinformatic Clinical Outcome Pathway confirmed that in vitro trophic effects of MYO could be transferred in vivo with high predictability, providing robust credence of its efficacy for oral health.ConclusionOur reverse bed-to-benchside data indicate that MYO might antagonize the detrimental effects of IR on tooth decay. This provides feasibility for clinical studies on MYO as a regenerative factor in dentistry and oral surgery, including dysmetabolic/aging conditions, bone reconstruction in oral destructive/necrotic disorders, dental implants, and for empowering the efficacy of a number of tissue engineering methodologies in dentistry and oral surgery

Tumor hypoxia does not drive differentiation of tumor-associated macrophages but rather fine-tunes the M2-like macrophage population

Tumor-associated macrophages (TAM) are exposed to multiple microenvironmental cues in tumors, which collaborate to endow these cells with protumoral activities. Hypoxia, caused by an imbalance in oxygen supply and demand because of a poorly organized vasculature, is often a prominent feature in solid tumors. However, to what extent tumor hypoxia regulates the TAM phenotype in vivo is unknown. Here, we show that the myeloid infiltrate in mouse lung carcinoma tumors encompasses two morphologically distinct CD11b(hi)F4/80(hi)Ly6C(lo) TAM subsets, designated as MHC-II(lo) and MHC-II(hi) TAM, both of which were derived from tumor-infiltrating Ly6C(hi) monocytes. MHC-II(lo) TAM express higher levels of prototypical M2 markers and reside in more hypoxic regions. Consequently, MHC-II(lo) TAM contain higher mRNA levels for hypoxia-regulated genes than their MHC-II(hi) counterparts. To assess the in vivo role of hypoxia on these TAM features, cancer cells were inoculated in prolyl hydroxylase domain 2 (PHD2)-haplodeficient mice, resulting in better-oxygenated tumors. Interestingly, reduced tumor hypoxia did not alter the relative abundance of TAM subsets nor their M2 marker expression, but specifically lowered hypoxia-sensitive gene expression and angiogenic activity in the MHC-II(lo) TAM subset. The same observation in PHD2(+/+) → PHD2(+/-) bone marrow chimeras also suggests organization of a better-oxygenized microenvironment. Together, our results show that hypoxia is not a major driver of TAM subset differentiation, but rather specifically fine-tunes the phenotype of M2-like MHC-II(lo) TAM

Analysis of human MDM4 variants in papillary thyroid carcinomas reveals new potential markers of cancer properties

A wild-type (wt) p53 gene characterizes thyroid tumors, except for the rare anaplastic histotype. Because p53 inactivation is a prerequisite for tumor development, alterations of p53 regulators represent an alternative way to impair p53 function. Indeed, murine double minute 2 (MDM2), the main p53 negative regulator, is overexpressed in many tumor histotypes including those of the thyroid. A new p53 regulator, MDM4 (a.k.a. MDMX or HDMX) an analog of MDM2, represents a new oncogene although its impact on tumor properties remains largely unexplored. We estimated levels of MDM2, MDM4, and its variants, MDM4-S (originally HDMX-S) and MDM4-211 (originally HDMX211), in a group of 57 papillary thyroid carcinomas (PTC), characterized by wt tumor protein 53, in comparison to matched contra-lateral lobe normal tissue. Further, we evaluated the association between expression levels of these genes and the histopathological features of tumors. Quantitative real-time polymerase chain reaction revealed a highly significant downregulation of MDM4 mRNA in tumor tissue compared to control tissue (P < 0.0001), a finding confirmed by western blot on a subset of 20 tissue pairs. Moreover, the tumor-to-normal ratio of MDM4 levels for each individual was significantly lower in late tumor stages, suggesting a specific downregulation of MDM4 expression with tumor progression. In comparison, MDM2 messenger RNA (mRNA) and protein levels were frequently upregulated with no correlation with MDM4 levels. Lastly, we frequently detected overexpression of MDM4-S mRNA and presence of the aberrant form, MDM4-211 in this tumor group. These findings indicate that MDM4 alterations are a frequent event in PTC. It is worthy to note that the significant downregulation of full-length MDM4 in PTC reveals a novel status of this factor in human cancer that counsels careful evaluation of its role in human tumorigenesis and of its potential as therapeutic target

ER Stress Responses: An Emerging Modulator for Innate Immunity

The endoplasmic reticulum (ER) is a critical organelle, storing the majority of calcium and governing protein translation. Thus, it is crucial to keep the homeostasis in all ER components and machineries. The ER stress sensor pathways, including IRE1/sXBP1, PERK/EIf2α and ATF6, orchestrate the major regulatory circuits to ensure ER homeostasis. The embryonic or postnatal lethality that occurs upon genetic depletion of these sensors reveals the essential role of the ER stress pathway in cell biology. In contrast, the impairment or excessive activation of ER stress has been reported to cause or aggravate several diseases such as atherosclerosis, diabetes, NAFDL/NASH, obesity and cancer. Being part of innate immunity, myeloid cells are the first immune cells entering the inflammation site. Upon entry into a metabolically stressed disease environment, activation of ER stress occurs within the myeloid compartment, leading to the modulation of their phenotype and functions. In this review, we discuss causes and consequences of ER stress activation in the myeloid compartment with a special focus on the crosstalk between ER, innate signaling and metabolic environments.</jats:p