Publications

[No abstract available]

JTD Keywords: Alpha synuclein, Animal cell, Article, Astrocyte, Brain blood flow, Capillary endothelial cell, Cardiovascular system, Cell interaction, Coculture, Degenerative disease, Differential expression analysis, Endothelium cell, Entactin, Extracellular matrix, Fibronectin, Gene expression, Human, Human cell, Huntington chorea, Hydroxyapatite, In vitro study, Induced pluripotent stem cell, Laminin, Macrophage, Maturity, Microglia, Nervous system, Nervous system inflammation, Neuroprotection, Neurotoxicity, Nonhuman, Parkinson disease, Pericyte, Perivascular space, Personalized medicine, Shear stress, Smooth muscle cell, Three dimensional printing

Ex vivo-loaded white blood cells (WBC) can transfer cargo to pathological foci in the central nervous system (CNS). Here we tested affinity ligand driven in vivo loading of WBC in order to bypass the need for ex vivo WBC manipulation. We used a mouse model of acute brain inflammation caused by local injection of tumor necrosis factor alpha (TNF-α). We intravenously injected nanoparticles targeted to intercellular adhesion molecule 1 (anti-ICAM/NP). We found that (A) at 2 h, >20% of anti-ICAM/NP were localized to the lungs; (B) of the anti-ICAM/NP in the lungs >90% were associated with leukocytes; (C) at 6 and 22 h, anti-ICAM/NP pulmonary uptake decreased; (D) anti-ICAM/NP uptake in brain increased up to 5-fold in this time interval, concomitantly with migration of WBCs into the injured brain. Intravital microscopy confirmed transport of anti-ICAM/NP beyond the blood-brain barrier and flow cytometry demonstrated complete association of NP with WBC in the brain (98%). Dexamethasone-loaded anti-ICAM/liposomes abrogated brain edema in this model and promoted anti-inflammatory M2 polarization of macrophages in the brain. In vivo targeted loading of WBC in the intravascular pool may provide advantages of coopting WBC predisposed to natural rapid mobilization from the lungs to the brain, connected directly via conduit vessels.

JTD Keywords: drug delivery, icam-1, inflammation, lung injury, messenger-rna, migration, model, nanoparticles, neutrophils, pharmacokinetics, t-cells, white bloodcells, Adhesion molecules, Brain, Drug delivery, Inflammation, Nanoparticles, Pharmacokinetics, White blood cells

Liver cirrhosis is the end stage of all chronic liver diseases and contributes significantly to overall mortality of 2% globally. The age-standardized mortality from liver cirrhosis in Europe is between 10 and 20% and can be explained by not only the development of liver cancer but also the acute deterioration in the patient's overall condition. The development of complications including accumulation of fluid in the abdomen (ascites), bleeding in the gastrointestinal tract (variceal bleeding), bacterial infections, or a decrease in brain function (hepatic encephalopathy) define an acute decompensation that requires therapy and often leads to acute-on-chronic liver failure (ACLF) by different precipitating events. However, due to its complexity and organ-spanning nature, the pathogenesis of ACLF is poorly understood, and the common underlying mechanisms leading to the development of organ dysfunction or failure in ACLF are still elusive. Apart from general intensive care interventions, there are no specific therapy options for ACLF. Liver transplantation is often not possible in these patients due to contraindications and a lack of prioritization. In this review, we describe the framework of the ACLF-I project consortium funded by the Hessian Ministry of Higher Education, Research and the Arts (HMWK) based on existing findings and will provide answers to these open questions.

JTD Keywords: 12/15-lipoxygenase, combination, inflammation, interleukin-22, metabolism, mortality, organ failure, portal-hypertension, receptor, regeneration, systemic inflammation, systems medicine, translational hepatology, Decompensated cirrhosis, Organ failure, Systemic inflammation, Systems medicine, Translational hepatology

Immunoassays show great potential for the detection of low levels of cytokines, due to their high sensitivity and excellent specificity. There is a particular demand for biosensors that enable both high-throughput screening and continuous monitoring of clinically relevant cytokines such as interleukin-6 (IL-6) and tumor necrosis factor-α (TNFα). To this end, we here introduce a novel bioluminescent immunoassay based on the ratiometric plug-and-play immunodiagnostics (RAPPID) platform, with an improved intrinsic signal-to-background and an >80-fold increase in the luminescent signal. The new dRAPPID assay, comprising a dimeric protein G adapter connected via a semiflexible linker, was applied to detect the secretion of IL-6 by breast carcinoma cells upon TNFα stimulation and the production of low concentrations of IL-6 (∼18 pM) in an endotoxin-stimulated human 3D muscle tissue model. Moreover, we integrated the dRAPPID assay in a newly developed microfluidic device for the simultaneous and continuous monitoring of changes in IL-6 and TNFα in the low-nanomolar range. The luminescence-based read-out and the homogeneous nature of the dRAPPID platform allowed for detection with a simple measurement setup, consisting of a digital camera and a light-sealed box. This permits the usage of the continuous dRAPPID monitoring chip at the point of need, without the requirement for complex or expensive detection techniques.

JTD Keywords: cells, code, elisa, il-6, inflammation, kits, pathogenesis, procalcitonin, release, Cytokines, Humans, Immunoassay, Immunologic tests, Interleukin-6, Tumor necrosis factor-alpha

Lafora disease is a rare disorder caused by loss of function mutations in either the EPM2A or NHLRC1 gene. The initial symptoms of this condition are most commonly epileptic seizures, but the disease progresses rapidly with dementia, neuropsychiatric symptoms, and cognitive deterioration and has a fatal outcome within 5–10 years after onset. The hallmark of the disease is the accumulation of poorly branched glycogen in the form of aggregates known as Lafora bodies in the brain and other tissues. Several reports have demonstrated that the accumulation of this abnormal glycogen underlies all the pathologic traits of the disease. For decades, Lafora bodies were thought to accumulate exclusively in neurons. However, it was recently identified that most of these glycogen aggregates are present in astrocytes. Importantly, astrocytic Lafora bodies have been shown to contribute to pathology in Lafora disease. These results identify a primary role of astrocytes in the pathophysiology of Lafora disease and have important implications for other conditions in which glycogen abnormally accumulates in astrocytes, such as Adult Polyglucosan Body disease and the buildup of Corpora amylacea in aged brains.

JTD Keywords: abnormal glycogen, accumulation, aggregation, bodies, branching enzyme deficiency, corpora-amylacea, epilepsy, glycogen, lafora disease, mice, mouse model, neurodegeneration, neuroinflammation, progressive myoclonus epilepsy, ubiquitin ligase, Glycogen, Neuroinflammation, Polyglucosan body disease

Microglia are very sensitive to changes in the environment and respond through morphological, functional and metabolic adaptations. To depict the modifications microglia undergo under healthy and pathological conditions, we developed free access image analysis scripts to quantify microglia morphologies and phagocytosis. Neuron-glia cultures, in which microglia express the reporter tdTomato, were exposed to excitotoxicity or excitotoxicity + inflammation and analysed 8 h later. Neuronal death was assessed by SYTOX staining of nucleus debris and phagocytosis was measured through the engulfment of SYTOX+ particles in microglia. We identified seven morphologies: round, hypertrophic, fried egg, bipolar and three 'inflamed' morphologies. We generated a classifier able to separate them and assign one of the seven classes to each microglia in sample images. In control cultures, round and hypertrophic morphologies were predominant. Excitotoxicity had a limited effect on the composition of the populations. By contrast, excitotoxicity + inflammation promoted an enrichment in inflamed morphologies and increased the percentage of phagocytosing microglia. Our data suggest that inflammation is critical to promote phenotypical changes in microglia. We also validated our tools for the segmentation of microglia in brain slices and performed morphometry with the obtained mask. Our method is versatile and useful to correlate microglia sub-populations and behaviour with environmental changes.

JTD Keywords: classification, identification, image analysis, injury, morphometry, neuroinflammation, neurotoxicity, phagocytosis, Classification, Image analysis, Microglia, Morphometry, Neuroinflammation, Nitric-oxide, Phagocytosis

Acute Respiratory Distress Syndrome is one of the more common fatal complications in COVID-19, characterized by a highly aberrant inflammatory response. Pre-clinical models to study the effect of cell therapy and anti-inflammatory treatments have not comprehensively reproduced the disease due to its high complexity. This work presents a novel physiomimetic in vitro model for Acute Respiratory Distress Syndrome using lung extracellular matrix-derived hydrogels and organ-on-a-chip devices. Monolayres of primary alveolar epithelial cells were cultured on top of decellullarized lung hydrogels containing primary lung mesenchymal stromal cells. Then, cyclic stretch was applied to mimic breathing, and an inflammatory response was induced by using a bacteriotoxin hit. Having simulated the inflamed breathing lung environment, we assessed the effect of an anti-inflammatory drug (i.e., dexamethasone) by studying the secretion of the most relevant inflammatory cytokines. To better identify key players in our model, the impact of the individual factors (cyclic stretch, decellularized lung hydrogel scaffold, and the presence of mesenchymal stromal cells) was studied separately. Results showed that developed model presented a more reduced inflammatory response than traditional models, which is in line with what is expected from the response commonly observed in patients. Further, from the individual analysis of the different stimuli, it was observed that the use of extracellular matrix hydrogels obtained from decellularized lungs had the most significant impact on the change of the inflammatory response. The developed model then opens the door for further in vitro studies with a better-adjusted response to the inflammatory hit and more robust results in the test of different drugs or cell therapy.

JTD Keywords: alveolar epithelial cells, ards, extracellular matrix, hydrogels, inflammation, lung-on-a-chip, Acute lung injury, Alveolar epithelial cells, Ards, Dexamethasone, Epithelial-mesenchymal transition, Extracellular matrix, Extracellular-matrix, Hydrogels, Inflammation, Lung-on-a-chip, Mesenchymal stromal cells, Oxygen, Stem-cells

3DNA holds promise as a carrier for drugs that can be intercalated into its core or linked to surface arms. Coupling 3DNA to an antibody targeting intercellular adhesion molecule 1 (ICAM-1) results in high lung-specific biodistributions in vivo. While the role of individual parameters on ICAM-1 targeting has been studied for other nanocarriers, it has never been examined for 3DNA or in a manner capable of revealing the hierarchic interplay among said parameters. In this study, we used 2-layer vs. 4-layer anti-ICAM 3DNA and radiotracing to examine biodistribution in mice. We found that, below saturating conditions and within the ranges tested, the density of targeting antibodies on 3DNA is the most relevant parameter driving lung targeting over liver clearance, compared to the number of antibodies per carrier, total antibody dose, 3DNA dose, 3DNA size, or the administered concentration, which influenced the dose in organs but not the lung specific-over-liver clearance ratio. Data predicts that lung-specific delivery of intercalating (core loaded) drugs can be tuned using this biodistribution pattern, while that of arm-linked (surface loaded) drugs requires a careful parametric balance because increasing anti-ICAM density reduces the number of 3DNA arms available for drug loading.

JTD Keywords: 3dna nanocarrier, acid sphingomyelinase, antibody, carrier design parameters, carriers, dna nanostructures, doxorubicin, drug type, icam-1, inflammation, lung targeting, multiparametric hierarchy, nanoparticles, size, 3dna nanocarrier, Intracellular delivery, Multiparametric hierarchy

Abstract Lafora disease is a fatal neurodegenerative childhood dementia caused by loss-of-function mutations in either the laforin or malin gene. The hallmark of the disease is the accumulation of abnormal glycogen aggregates known as Lafora bodies (LBs) in the brain and other tissues. These aggregates are responsible for the pathological features of the disease. As a monogenic disorder, Lafora disease is a good candidate for gene therapy-based approaches. However, most patients are diagnosed after the appearance of the first symptoms and thus when LBs are already present in the brain. In this context, it was not clear whether the restoration of a normal copy of the defective gene (either laforin or malin) would prove effective. Here we evaluated the effect of restoring malin in a malin-deficient mouse model of Lafora disease as a proof of concept for gene replacement therapy. To this end, we generated a malin-deficient mouse in which malin expression can be induced at a certain time. Our results reveal that malin restoration at an advanced stage of the disease arrests the accumulation of LBs in brain and muscle, induces the degradation of laforin and glycogen synthase bound to the aggregates, and ameliorates neuroinflammation. These results identify malin restoration as the first therapeutic strategy to show effectiveness when applied at advanced stages of Lafora disease.

JTD Keywords: accumulation, gene therapy, glycogen, lafora disease, neurodegeneration, neuroinflammation, neurons, targets, Carbohydrate-binding domain, Glycogen, Neuroinflammation

Heterotopic ossification (HO) is a condition triggered by an injury leading to the formation of mature lamellar bone in extraskeletal soft tissues. Despite being a frequent complication of orthopedic and trauma surgery, brain and spinal injury, the etiology of HO is poorly understood. The aim of this study is to evaluate the hypothesis that a sustained local ionic homeostatic imbalance (SLIHI) created by mineral formation during tissue calcification modulates inflammation to trigger HO. This evaluation also considers the role SLIHI could play for the design of cell-free, drug-free osteoinductive bone graft substitutes. The evaluation contains five main sections. The first section defines relevant concepts in the context of HO and provides a summary of proposed causes of HO. The second section starts with a detailed analysis of the occurrence and involvement of calcification in HO. It is followed by an explanation of the causes of calcification and its consequences. This allows to speculate on the potential chemical modulators of inflammation and triggers of HO. The end of this second section is devoted to in vitro mineralization tests used to predict the ectopic potential of materials. The third section reviews the biological cascade of events occurring during pathological and material-induced HO, and attempts to propose a quantitative timeline of HO formation. The fourth section looks at potential ways to control HO formation, either acting on SLIHI or on inflammation. Chemical, physical, and drug-based approaches are considered. Finally, the evaluation finishes with a critical assessment of the definition of osteoinduction.

JTD Keywords: apatite, beta-tricalcium phosphate, bone, bone graft, bone morphogenetic protein, demineralized bone-matrix, experimental myositis-ossificans, extracellular calcium, heterotopic ossification, in-vitro, inflammation, multinucleated giant-cells, osteoinduction, spinal-cord-injury, total hip-arthroplasty, traumatic brain-injury, Apatite, Calcium-sensing receptor, Osteoinduction

Albumin has been used primarily as a plasma expander, since it leads to an increase in the circulating blood volume. Current generally recommended indications for albumin therapy in cirrhotic patients are the prevention of circulatory dysfunction after large-volume paracentesis, the prevention of hepatorenal syndrome (HRS) in patients with spontaneous bacterial peritonitis (SBP), and the management of HRS in combination with vasoconstrictors. Yet, new indications for albumin have been tested in the recent years and are outlined in this short review.New data show that albumin both supports the circulation and reduces systemic inflammation. In addition, to its oncotic function, it acts as an antioxidant, radical scavenger, and immune modulator. These nononcotic properties explain why long-term albumin administration in patients with decompensated cirrhosis may be useful in the prevention of associated complications (acute-on-chronic liver failure, infections). New data show that long-term albumin therapy in patients with cirrhosis and ascites improves survival, prevents complications, simplifies ascites management, and lowers hospitalization rates. The so-called disease-modifying effects of long-term albumin therapy may have a favorable effect on the course of the disease. Nevertheless, the optimal dosage and administration intervals have not yet been finally defined.Albumin therapy is effective in the indications already recommended by the guidelines. A possible extension of the indication for albumin administration in non-SBP infections and as long-term therapy is promising, but should be confirmed by further studies.Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.

JTD Keywords: ascites, failure, hepatorenal syndrome, hospitalized-patients, hypothesis, infections, portal hypertension, spontaneous bacterial peritonitis, systemic inflammation, Acute-on-chronic liver failure, Human serum-albumin

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JTD Keywords: cancer, cells, cellular basis, delivery, encapsulation, in-vitro, inflammation, macrophage, methotrexate, pathogenesis, polymersome, polymersomes, synoviocyte, Arthritis, Rheumatoid-arthritis

The use of silver nanoparticles (Ag NPs) in the biomedical field deserves a mindful analysis of the possible inflammatory response which could limit their use in the clinic. Despite the anti-cancer properties of Ag NPs having been widely demonstrated, there are still few studies concerning their involvement in the activation of specific inflammatory pathways. The inflammatory outcome depends on the synthetic route used in the NPs production, in which toxic reagents are employed. In this work, we compared two types of Ag NPs, obtained by two different chemical routes: conventional synthesis using sodium citrate and a green protocol based on leaf extracts as a source of reduction and capping agents. A careful physicochemical characterization was carried out showing spherical and stable Ag NPs with an average size between 20 nm and 35 nm for conventional and green Ag NPs respectively. Then, we evaluated their ability to induce the activation of inflammation in Human Leukemic Monocytes (THP-1) differentiated into M0 macrophages using 1 µM and 2 µM NPs concentrations (corresponded to 0.1 µg/mL and 0.2 µg/mL respectively) and two-time points (24 h and 48 h). Our results showed a clear difference in Nuclear Factor ?B (NF-?b) activation, Interleukins 6–8 (IL-6, IL-8) secretion, Tumor Necrosis Factor-? (TNF-?) and Cyclooxygenase-2 (COX-2) expression exerted by the two kinds of Ag NPs. Green Ag NPs were definitely tolerated by macrophages compared to conventional Ag NPs which induced the activation of all the factors mentioned above. Subsequently, the exposure of breast cancer cell line (MCF-7) to the green Ag NPs showed that they exhibited antitumor activity like the conventional ones, but surprisingly, using the MCF-10A line (not tumoral breast cells) the green Ag NPs did not cause a significant decrease in cell viability. © 2022 by the authors. Licensee MDPI, Basel, Switzerland.

JTD Keywords: activation, biosynthesis, gold nanoparticles, green route, inflammation response, mechanism, metal, nanotechnology, physico-chemical properties, raman-spectroscopy, resonance, silver nanoparticles, surface, Biomedical fields, Cell culture, Cell death, Chemical activation, Chemical routes, Conventional synthesis, Diseases, Green route, Inflammation response, Inflammatory response, Macrophages, Metal nanoparticles, Nf-kappa-b, Pathology, Physico-chemical properties, Physicochemical property, Property, Silver nanoparticles, Sodium compounds, Synthetic routes, Toxic reagents

Lafora disease (LD) is a fatal childhood-onset dementia characterized by the extensive accumulation of glycogen aggregates—the so-called Lafora Bodies (LBs)—in several organs. The accumulation of LBs in the brain underlies the neurological phenotype of the disease. LBs are composed of abnormal glycogen and various associated proteins, including p62, an autophagy adaptor that participates in the aggregation and clearance of misfolded proteins. To study the role of p62 in the formation of LBs and its participation in the pathology of LD, we generated a mouse model of the disease (malinKO) lacking p62. Deletion of p62 prevented LB accumulation in skeletal muscle and cardiac tissue. In the brain, the absence of p62 altered LB morphology and increased susceptibility to epilepsy. These results demonstrate that p62 participates in the formation of LBs and suggest that the sequestration of abnormal glycogen into LBs is a protective mechanism through which it reduces the deleterious consequences of its accumulation in the brain. © 2021, The Author(s).

JTD Keywords: accumulation, astrocytes, autophagy receptors, contributes, deficient mice, epilepsy, glycogen, lafora bodies, lafora disease, malin, metabolism, neurodegeneration, neuroinflammation, p62, polyglucosan bodies, temporal-lobe epilepsy, Epilepsy, Glycogen, Inclusion-body formation, Lafora bodies, Lafora disease, Malin, Neuroinflammation, P62

Unlike the attachment of soft epithelial skin tissue to penetrating solid natural structures like fingernails and teeth, sealing around percutaneous/permucosal devices such as dental implants is hindered by inflammation and epidermal down growth. Here, we employed a dual keratinocyte-adhesive peptide and anti-inflammatory biomolecule coating on titanium to promote oral epithelial tissue attachment. For minimizing inflammation-triggered epidermal down growth, we coated pristine and oxygen plasma pre-treated polished titanium (pTi) with conjugated linoleic acid (CLA). Further, in order to aid in soft tissue attachment via the formation of hemidesmosomes, adhesive structures by oral keratinocytes, we coated the anionic linoleic acid (LA) adsorbed titanium with cationic cell adhesive peptides (CAP), LamLG3, a peptide derived from Laminin 332, the major extracellular matrix component of the basement membrane in skin tissue and Net1, derived from Netrin-1, a neural chemoattractant capable of epithelial cell attachment via alpha 6 beta 4 integrins. The dual CLA-CAP coatings on pTi were characterized by X-ray photoelectron spectroscopy and dynamic water contact angle measurements. The proliferation of human oral keratinocytes (TERT-2/OKF6) was accelerated on the peptide coated titanium while also promoting the expression of Col XVII and beta-4 integrin, two markers for hemidesmosomes. Simultaneously, CLA coating suppressed the production of inducible nitric oxide synthase (anti-iNOS); a pro-inflammatory M1 marker expressed in lipopolysaccharide (LPS) stimulated murine macrophages (RAW 264.7) and elevated expression of anti-CD206, associated to an anti-inflammatory M2 macrophage phenotype. Taken together, the dual keratinocyte-adhesive peptide and anti-inflammatory biomolecule coating on titanium can help reduce inflammation and promote permucosal/peri-implant soft tissue sealing.

JTD Keywords: Adhesives, Animal, Animals, Anti-inflammatories, Anti-inflammatory agents, Antiinflammatory agent, Biomolecules, Bone, Cell adhesion, Cell-adhesives, Coatings, Conjugated linoleic acid, Conjugated linoleic-acid, Contact angle, Hemidesmosome, Hemidesmosomes, Human, Humans, Hydroxyapatite, Inflammation, Integrins, Keratinocyte, Keratinocytes, Linoleic acid, Macrophages, Mice, Mouse, Nitric oxide, Oral implants, Pathology, Peptides, Skin tissue, Soft tissue, Supplementation, Surface properties, Surface property, Tissue, Titania, Titanium, X ray photoelectron spectroscopy

Despite tremendous progress in the understanding of COVID-19, mechanistic insight into immunological, disease-driving factors remains limited. We generated maVie16, a mouse-adapted SARS-CoV-2, by serial passaging of a human isolate. In silico modeling revealed how only three Spike mutations of maVie16 enhanced interaction with murine ACE2. maVie16 induced profound pathology in BALB/c and C57BL/6 mice, and the resulting mouse COVID-19 (mCOVID-19) replicated critical aspects of human disease, including early lymphopenia, pulmonary immune cell infiltration, pneumonia, and specific adaptive immunity. Inhibition of the proinflammatory cyto-kines IFN? and TNF substantially reduced immunopathology. Importantly, genetic ACE2-deficiency completely prevented mCOVID-19 development. Finally, inhalation therapy with recombinant ACE2 fully protected mice from mCOVID-19, revealing a novel and efficient treatment. Thus, we here present maVie16 as a new tool to model COVID-19 for the discovery of new therapies and show that disease severity is determined by cytokine-driven immunopathology and critically dependent on ACE2 in vivo. © Gawish et al.

JTD Keywords: covid-19 mouse model, covid-19 therapy, cytokine storm, immunology, inflammation, mavie16, mouse, mouse-adapted sars-cov-2, program, recombinant soluble ace2, tmprss2, Adaptive immunity, Angiotensin converting enzyme 2, Angiotensin-converting enzyme 2, Animal, Animal cell, Animal experiment, Animal model, Animal tissue, Animals, Apoptosis, Article, Bagg albino mouse, Breathing rate, Bronchoalveolar lavage fluid, C57bl mouse, Cell composition, Cell infiltration, Controlled study, Coronavirus disease 2019, Coronavirus spike glycoprotein, Covid-19, Cytokeratin 18, Cytokine production, Dipeptidyl carboxypeptidase, Disease model, Disease models, animal, Disease severity, Drosophila-melanogaster, Enzyme linked immunosorbent assay, Expression vector, Flow cytometry, Gamma interferon, Gene editing, Gene expression, Gene mutation, Genetic engineering, Genetics, Glycosylation, High mobility group b1 protein, Histology, Histopathology, Immune response, Immunocompetent cell, Immunology, Immunopathology, Interferon-gamma, Interleukin 2, Metabolism, Mice, inbred balb c, Mice, inbred c57bl, Mouse-adapted sars-cov-2, Myeloperoxidase, Neuropilin 1, Nonhuman, Nucleocapsid protein, Pathogenicity, Peptidyl-dipeptidase a, Pyroptosis, Recombinant soluble ace2, Renin angiotensin aldosterone system, Rna extraction, Rna isolation, Sars-cov-2, Severe acute respiratory syndrome coronavirus 2, Spike glycoprotein, coronavirus, T lymphocyte activation, Trabecular meshwork, Tumor necrosis factor, Virology, Virus load, Virus replication, Virus transmission, Virus virulence

Sex differences in immune-mediated diseases are linked to the activity of estrogens on innate immunity cells, including macrophages. Tamoxifen (TAM) is a selective estrogen receptor modulator (SERM) used in estrogen receptor-alpha (ER alpha)-dependent breast cancers and off-target indications such as infections, although the immune activity of TAM and its active metabolite, 4-OH tamoxifen (4HT), is poorly characterized. Here, we aimed at investigating the endocrine and immune activity of these SERMs in macrophages. Using primary cultures of female mouse macrophages, we analyzed the expression of immune mediators and activation of effector functions in competition experiments with SERMs and 17 beta-estradiol (E2) or the bacterial endotoxin LPS. We observed that 4HT and TAM induce estrogen antagonist effects when used at nanomolar concentrations, while pharmacological concentrations that are reached by TAM in clinical settings regulate the expression of VEGF alpha and other immune activation genes by ER alpha- and G protein-coupled receptor 1 (GPER1)-independent mechanisms that involve NRF2 through PI3K/Akt-dependent mechanisms. Importantly, we observed that SERMs potentiate cell phagocytosis and modify the effects of LPS on the expression of inflammatory cytokines, such as TNF alpha and IL1 beta, with an overall increase in cell inflammatory phenotype, further sustained by potentiation of IL1 beta secretion through caspase-1 activation.

JTD Keywords: drug repurposing, inflammation, macrophage, nrf2, Apoptosis, Breast-cancer, Drug repurposing, Expression, Inflammation, Macrophage, Nrf2, Resistance, Sex-differences, Tamoxifen, Tumor-associated macrophages

The hallmark of Lafora disease, a fatal neurodegenerative disorder, is the accumulation of intracellular glycogen aggregates called Lafora bodies. Until recently, it was widely believed that brain Lafora bodies were present exclusively in neurons and thus that Lafora disease pathology derived from their accumulation in this cell population. However, recent evidence indicates that Lafora bodies are also present in astrocytes. To define the role of astrocytic Lafora bodies in Lafora disease pathology, we deleted glycogen synthase specifically from astrocytes in a mouse model of the disease (malin(KO)). Strikingly, blocking glycogen synthesis in astrocytes-thus impeding Lafora bodies accumulation in this cell type-prevented the increase in neurodegeneration markers, autophagy impairment, and metabolic changes characteristic of the malin(KO) model. Conversely, mice that over-accumulate glycogen in astrocytes showed an increase in these markers. These results unveil the deleterious consequences of the deregulation of glycogen metabolism in astrocytes and change the perspective that Lafora disease is caused solely by alterations in neurons.

JTD Keywords: Bodies, Deficient mice, Epilepsy, Glycogen, Impairment, Lafora disease, Malin, Modulation, Mouse model, Neurodegeneration, Neuroinflammation, Neurons, Progressive myoclonus epilepsy, Seizure susceptibility, Synthase

Background: In recent decades, gold nanoparticle (Au NP)-based cancer therapy has been heavily debated. The physico-chemical properties of AuNPs can be exploited in photothermal therapy, making them a powerful tool for selectively killing cancer cells. However, the synthetic side products and capping agents often induce a strong activation of the inflammatory pathways of macrophages, thus limiting their further applications in vivo. Methods: Here, we described a green method to obtain stable polyphenol-capped AuNPs (Au NPs@polyphenols), as polyphenols are known for their anti-inflammatory and anticancer properties. These NPs were used in human macrophages to test key inflammation-related markers, such as NF-κB, TNF-α, and interleukins-6 and 8. The results were compared with similar NPs obtained by a traditional chemical route (without the polyphenol coating), proving the potential of Au NPs@polyphenols to strongly promote the shutdown of inflammation. This was useful in developing them for use as heat-synergized tools in the thermal treatment of two types of cancer cells, namely, breast cancer (MCF-7) and neuroblastoma (SH-SY5Y) cells. The cell viability, calcium release, oxidative stress, HSP-70 expression, mitochondrial, and DNA damage, as well as cytoskeleton alteration, were evaluated. Results: Our results clearly demonstrate that the combined strategy markedly exerts anticancer effects against the tested cancer cell, while neither of the single treatments (only heat or only NPs) induced significant changes. Conclusions: Au NP@polyphenols may be powerful agents in cancer treatment.

JTD Keywords: antioxidant, aunps, biocompatibility, biology, calcium, cancer, green synthesis, inflammation response, inhibition, interleukin-6, mechanisms, natural polyphenols, physico-chemical properties, polyphenols, size, thermal treatment, Aunps, Cancer, Green synthesis, Inflammation response, Nobilis l. leaves, Physico-chemical properties, Polyphenols, Thermal treatment

© 2020 Elsevier Ltd Central nervous system (CNS) injuries do not heal properly in contrast to normal tissue repair, in which functional recovery typically occurs. The reason for this dichotomy in wound repair is explained in part by macrophage and microglial malfunction, affecting both the extrinsic and intrinsic barriers to appropriate axonal regeneration. In normal healing tissue, macrophages promote the repair of injured tissue by regulating transitions through different phases of the healing response. In contrast, inflammation dominates the outcome of CNS injury, often leading to secondary damage. Therefore, an understanding of the molecular mechanisms underlying this dichotomy is critical to advance in neuronal repair therapies. Recent studies highlight the plasticity and complexity of macrophages and microglia beyond the classical view of the M1/M2 polarization paradigm. This plasticity represents an in vivo continuous spectrum of phenotypes with overlapping functions and markers. Moreover, macrophage and microglial plasticity affect many events essential for neuronal regeneration after injury, such as myelin and cell debris clearance, inflammation, release of cytokines, and trophic factors, affecting both intrinsic neuronal properties and extracellular matrix deposition. Until recently, this complexity was overlooked in the translation of therapies modulating these responses for the treatment of neuronal injuries. However, recent studies have shed important light on the underlying molecular mechanisms of this complexity and its transitions and effects on regenerative events. Here we review the complexity of macrophages and microglia after neuronal injury and their roles in regeneration, as well as the underlying molecular mechanisms, and we discuss current challenges and future opportunities for treatment.

JTD Keywords: chemokines and cytokines, macrophages, microglia, neuroinflammation, neuronal injury, regeneration, Chemokines and cytokines, Macrophages, Microglia, Neuroinflammation, Neuronal injury, Regeneration

© 2020, Springer Nature Limited. The human gut microbiome has emerged as a major player in human health and disease. The liver, as the first organ to encounter microbial products that cross the gut epithelial barrier, is affected by the gut microbiome in many ways. Thus, the gut microbiome might play a major part in the development of liver diseases. The common end stage of liver disease is decompensated cirrhosis and the further development towards acute-on-chronic liver failure (ACLF). These conditions have high short-term mortality. There is evidence that translocation of components of the gut microbiota, facilitated by different pathogenic mechanisms such as increased gut epithelial permeability and portal hypertension, is an important driver of decompensation by induction of systemic inflammation, and thereby also ACLF. Elucidating the role of the gut microbiome in the aetiology of decompensated cirrhosis and ACLF deserves further investigation and improvement; and might be the basis for development of diagnostic and therapeutic strategies. In this Review, we focus on the possible pathogenic, diagnostic and therapeutic role of the gut microbiome in decompensation of cirrhosis and progression to ACLF.

JTD Keywords: albumin, decreases intestinal permeability, hepatic-encephalopathy, portal-vein thrombosis, rifaximin improves, secondary bile-acids, systemic inflammation, translocation, venous-pressure gradient, Spontaneous bacterial peritonitis

Alcoholism is one of the leading and increasingly prevalent reasons of liver associated morbidity and mortality worldwide. Alcoholic hepatitis (AH) constitutes a severe disease with currently no satisfying treatment options. Lipoxin A4 (LXA4), a 15-lipoxygenase (ALOX15)-dependent lipid mediator involved in resolution of inflammation, showed promising pre-clinical results in the therapy of several inflammatory diseases. Since inflammation is a main driver of disease progression in alcoholic hepatitis, we investigated the impact of endogenous ALOX15-dependent lipid mediators and exogenously applied LXA4 on AH development. A mouse model for alcoholic steatohepatitis (NIAAA model) was tested in Alox12/15+/+ and Alox12/15−/− mice, with or without supplementation of LXA4. Absence of Alox12/15 aggravated parameters of liver disease, increased hepatic immune cell infiltration in AH, and elevated systemic neutrophils as a marker for systemic inflammation. Interestingly, i.p. injections of LXA4 significantly lowered transaminase levels only in Alox12/15−/− mice and reduced hepatic immune cell infiltration as well as systemic inflammatory cytokine expression in both genotypes, even though steatosis progressed. Thus, while LXA4 injection attenuated selected parameters of disease progression in Alox12/15−/− mice, its beneficial impact on immunity was also apparent in Alox12/15+/+ mice. In conclusion, pro-resolving lipid mediators may be beneficial to reduce inflammation in alcoholic hepatitis.

JTD Keywords: Alcoholic hepatitis, Arachidonate 12/15-lipoxygenase (Alox12/15), Lipoxin A4, Resolution of inflammation, Specialized pro-resolving lipid mediators (SPMs)

Background & Aims: Acute-on-chronic liver failure (ACLF) is characterized by high short-term mortality and systemic inflammation (SI). Recently, different cardiodynamic states were shown to independently predict outcomes in cirrhosis. The relationship between cardiodynamic states, SI, and portal hypertension and their impact on ACLF development remains unclear. The aim of this study was therefore to evaluate the interplay of cardiodynamic state and SI on fatal ACLF development in cirrhosis. Results: At inclusion, hemodynamic measures including cardiac index (CI) and hepatic venous pressure gradient of 208 patients were measured. Patients were followed prospectively for fatal ACLF development (primary endpoint). SI was assessed by proinflammatory markers such as interleukins (ILs) 6 and 8 and soluble IL-33 receptor (sIL-33R). Patients were divided according to CI (<3.2; 3.2-4.2; >4.2 L/min/m2) in hypo- (n = 84), normo- (n = 69) and hyperdynamic group (n = 55). After a median follow-up of 3 years, the highest risk of fatal ACLF was seen in hyperdynamic (35%) and hypodynamic patients (25%) compared with normodynamic (14%) (P = .011). Hyperdynamic patients showed the highest rate of SI. The detectable level of IL-6 was an independent predictor of fatal ACLF development. Conclusions: Cirrhotic patients with hyperdynamic and hypodynamic circulation have a higher risk of fatal ACLF. Therefore, the cardiodynamic state is strongly associated with SI, which is an independent predictor of development of fatal ACLF.

JTD Keywords: Acute-on-chronic liver failure, Circulation, Cirrhosis, Hemodynamic, Inflammation

Declining life expectancy and increasing all-cause mortality in the United States have been associated with unhealthy behaviors, socioecological factors, and preventable disease. A growing body of basic science, clinical research, and population health evidence points to the benefits of healthy behaviors, environments and policies to maintain health and prevent, treat, and reverse the root causes of common chronic diseases. Similarly, innovations in research methodologies, standards of evidence, emergence of unique study cohorts, and breakthroughs in data analytics and modeling create new possibilities for producing biomedical knowledge and clinical translation. To understand these advances and inform future directions research, The Lifestyle Medicine Research Summit was convened at the University of Pittsburgh on December 4–5, 2019. The Summit's goal was to review current status and define research priorities in the six core areas of lifestyle medicine: plant-predominant nutrition, physical activity, sleep, stress, addictive behaviors, and positive psychology/social connection. Forty invited subject matter experts (1) reviewed existing knowledge and gaps relating lifestyle behaviors to common chronic diseases, such as cardiovascular disease, diabetes, many cancers, inflammatory- and immune-related disorders and other conditions; and (2) discussed the potential for applying cutting-edge molecular, cellular, epigenetic and emerging science knowledge and computational methodologies, research designs, and study cohorts to accelerate clinical applications across all six domains of lifestyle medicine. Notably, federal health agencies, such as the Department of Defense and Veterans Administration have begun to adopt “whole-person health and performance” models that address these lifestyle and environmental root causes of chronic disease and associated morbidity, mortality, and cost. Recommendations strongly support leveraging emerging research methodologies, systems biology, and computational modeling in order to accelerate effective clinical and population solutions to improve health and reduce societal costs. New and alternative hierarchies of evidence are also be needed in order to assess the quality of evidence and develop evidence-based guidelines on lifestyle medicine. Children and underserved populations were identified as prioritized groups to study. The COVID-19 pandemic, which disproportionately impacts people with chronic diseases that are amenable to effective lifestyle medicine interventions, makes the Summit's findings and recommendations for future research particularly timely and relevant.

JTD Keywords: Chronic disease, Epigenetics, In silico modeling, Inflammation, Lifestyle medicine, Nutrition, Physical activity, Research methodologies

The macrophage-inducible C-type lectin (mincle) is part of the innate immune system and acts as a pattern recognition receptor for pathogen-associated molecular patterns (PAMPS) and damage-associated molecular patterns (DAMPs). Ligand binding induces mincle activation which consequently interacts with the signaling adapter Fc receptor, SYK, and NF-kappa-B. There is also evidence that mincle expressed on macrophages promotes intestinal barrier integrity. However, little is known about the role of mincle in hepatic fibrosis, especially in more advanced disease stages. Mincle expression was measured in human liver samples from cirrhotic patients and donors collected at liver transplantation and in patients undergoing bariatric surgery. Human results were confirmed in rodent models of cirrhosis and acute-on-chronic liver failure (ACLF). In these models, the role of mincle was investigated in liver samples as well as in peripheral blood monocytes (PBMC), tissues from the kidney, spleen, small intestine, and heart. Additionally, mincle activation was stimulated in experimental non-alcoholic steatohepatitis (NASH) by treatment with mincle agonist trehalose-6,6-dibehenate (TDB). In human NASH, mincle is upregulated with increased collagen production. In ApoE deficient mice fed high-fat western diet (NASH model), mincle activation significantly increases hepatic collagen production. In human cirrhosis, mincle expression is also significantly upregulated. Furthermore, mincle expression is associated with the stage of chronic liver disease. This could be confirmed in rat models of cirrhosis and ACLF. ACLF was induced by LPS injection in cirrhotic rats. While mincle expression and downstream signaling via FC receptor gamma, SYK, and NF-kappa-B are upregulated in the liver, they are downregulated in PBMCs of these rats. Although mincle expressed on macrophages might be beneficial for intestinal barrier integrity, it seems to contribute to inflammation and fibrosis once the intestinal barrier becomes leaky in advanced stages of chronic liver disease.

JTD Keywords: ACLF, Bacterial translocation, Fibrosis, Inflammation, NASH

Biomaterial implantation triggers inflammatory reactions. Understanding the effect of physicochemical features of biomaterials on the release of inflammatory cytokines from immune cells would be of great interest in view of designing bone graft materials to enhance the healing of bone defects. The present work investigated the interactions of two chemically and texturally different calcium phosphate (CaPs) substrates with macrophages, one of the main innate immune cells, and its further impact on osteogenic differentiation of bone forming cells. The behaviour of macrophages seeded on biomimetic calcium deficient hydroxyapatite (CDHA) and sintered β-tricalcium phosphate (β-TCP) was assessed in terms of the release of inflammatory cytokines and osteoclastogenic factors. The osteogenic differentiation of bone progenitor cells (bone marrow stromal cells (BMSCs) and osteoblastic cell line (SaOS-2)) were subsequently studied by incubating with the conditioned medium induced by macrophage-CaPs interaction in order to reveal the effect of immune cell reaction to CaPs on osteogenic differentiation. It was found that the incubation of macrophages with CaPs substrates caused a decrease of pro-inflammatory cytokines, more pronounced for β-TCP compared with CDHA showing significantly decreased IL-6, TNF-a, and iNOS. However, the macrophage-CDHA interaction resulted in a more favourable environment for osteogenic differentiation of osteoblasts with more collagen type I production and osteogenic genes (Runx2, BSP) expression, suggesting that osteogenic differentiation of bone cells is not only determined by the nature of biomaterials, but also significantly influenced by the inflammatory environment generated by the interaction of immune cells and biomaterials. Statement of Significance: The field of osteoimmunology highlights the importance of the cross-talk between immune and bone cells for effective bone regeneration. This tight interaction opens the door to new strategies that encompass the development of smart cell-instructive biomaterials which performance covers the events from early inflammation to osteogenesis. The present work links the anti-inflammatory and osteoimmunomodulatory features of synthetic bone grafts to their chemistry and texture, focussing on the cross-talk between macrophages and two major orchestrators of bone healing, namely primary mesenchymal stem cells and osteoblasts. The results emphasize the importance of the microenvironment created through the interaction between the substrate and the immune cells as it can stimulate osteogenic events and subsequently foster bone healing.

JTD Keywords: Calcium phosphates, Immunomodulation, Inflammation, Osteogenesis, Osteoimmunomodulation

Background: Acquired dysfunctional immunity in cirrhosis predisposes patients to frequent bacterial infections, especially spontaneous bacterial peritonitis (SBP), leading to systemic inflammation that is associated with poor outcome. But systemic inflammation can also be found in the absence of a confirmed infection. Detection of bacterial DNA has been investigated as a marker of SBP and as a predictor of prognosis. Data is, however, contradictory. Here we investigated whether levels of IL-6 and IL-8 putatively produced by myeloid cells in ascites are associated with systemic inflammation and whether inflammation depends on the presence of specific bacterial DNA. Methods and Materials: We enrolled 33 patients with decompensated liver cirrhosis from whom we collected paired samples of blood and ascites. IL-6 and IL-8 were measured in serum samples of all patients using ELISA. In a subset of 10 representative patients, bacterial DNA was extracted from ascites and whole blood, followed by 16S rRNA gene amplicon sequencing. Results: There were significantly higher levels of IL-6 in ascites fluid compared to blood samples in all patients. Interestingly, IL-6 levels in blood correlated tightly with disease severity and surrogates of systemic inflammation, while IL-6 levels in ascites did not. Moreover, patients with higher blood CRP levels showed greater SBP prevalence compared to patients with lower levels, despite similar positive culture results. Bacterial richness was also significantly higher in ascites compared to the corresponding patient blood. We identified differences in microbial composition and diversity between ascites and blood, but no tight relationship with surrogates of systemic inflammation could be observed. Discussion: In decompensated cirrhosis, markers of systemic inflammation and microbiota composition seem to be dysregulated in ascites and blood. While a relationship between systemic inflammation and microbiota composition seems to exist in blood, this is not the case for ascites in our hands. These data may suggest compartmentalization of the immune response and interaction of the latter with the microbiota especially in the blood compartment.

JTD Keywords: Acute-on-chronic liver failure, Ascites, Cirrhosis, Cytokines, Microbiome, Myeloid cells, Systemic inflammation

In the present work curcumin loaded hyalurosomes were proposed as innovative systems for the treatment of rheumatoid arthritis. Vesicles were prepared using a one-step and environmentally friendly method. Aiming at finding the most suitable formulation in terms of size, surface charge and stability on storage, an extensive pre-formulation study was performed using different type and amount of phospholipids. Curcumin loaded vesicles prepared with 180 mg/ml of Phospholipon 90G (P90G) and immobilized with sodium hyaluronate (2 mg/ml) were selected because of their small size (189 nm), homogeneous dispersion (PI 0.24), negative charge (−35 mV), suitable ability to incorporate high amount of curcumin (E% 88%) and great stability on storage. The in vitro study using fibroblast-like synovial cells cultured in synovial fluid, demonstrated the ability of these vesicles to downregulate the production of anti-apoptotic proteins IAP1 and IAP2 and stimulate the production of IL-10, while the production of IL-6 and IL-15 and reactive oxygen species was reduced, confirming their suitability in counteracting pathogenesis of rheumatoid arthritis.

JTD Keywords: Curcumin, IL-6 and IL-15, In vitro inflammation, Oxidative stress, Phospholipid vesicles, Synoviocytes

Glucocorticoid (GC) drugs are the cornerstone therapy used in the treatment of inflammatory diseases. Here, we report pH responsive poly(2-methacryloyloxyethyl phosphorylcholine)–poly(2-(diisopropylamino)ethyl methacrylate) (PMPC–PDPA) polymersomes as a suitable nanoscopic carrier to precisely and controllably deliver GCs within inflamed target cells. The in vitro cellular studies revealed that polymersomes ensure the stability, selectivity and bioavailability of the loaded drug within macrophages. At molecular level, we tested key inflammation-related markers, such as the nuclear factor-κB, tumour necrosis factor-α, interleukin-1β, and interleukin-6. With this, we demonstrated that pH responsive polymersomes are able to enhance the anti-inflammatory effect of loaded GC drug. Overall, we prove the potential of PMPC–PDPA polymersomes to efficiently promote the inflammation shutdown, while reducing the well-known therapeutic limitations in GC-based therapy.

JTD Keywords: Inflammation, Macrophages, Glucocorticoid, Polymersomes

The immobilization of natural molecules on synthetic bone grafts stands as a strategy to enhance their biological interactions. During the early stages of healing, immune cells and osteoclasts (OC) modulate the inflammatory response and resorb the biomaterial, respectively. In this study, heparin, a naturally occurring molecule in the bone extracellular matrix, was covalently immobilized on biomimetic calcium‐deficient hydroxyapatite (CDHA). The effect of heparin‐functionalized CDHA on inflammation and osteoclastogenesis was investigated using primary human cells and compared with pristine CDHA and beta-tricalcium phosphate (β-TCP). Biomimetic substrates led to lower oxidative stresses by neutrophils and monocytes than sintered β-TCP, even though no further reduction was induced by the presence of heparin. In contrast, heparinized CDHA fostered osteoclastogenesis. Optical images of stained TRAP positive cells showed an earlier and higher presence of multinucleated cells, compatible with OC at 14 days, while pristine CDHA and β-TCP present OC at 21–28 days. Although no statistically significant differences were found in the OC activity, microscopy images evidenced early stages of degradation on heparinized CDHA, compatible with osteoclastic resorption. Overall, the results suggest that the functionalization with heparin fostered the formation and activity of OC, thus offering a promising strategy to integrate biomaterials in the bone remodelling cycle by increasing their OC-mediated resorption.

JTD Keywords: Biomaterial, Heparin, Hydroxyapatite, Inflammation, Osteoclastogenesis

Immune cells are sensitive to the microstructural and textural properties of materials. Tuning the structural features of synthetic bone grafts could be a valuable strategy to regulate the specific response of the immune system, which in turn modulates the activity of bone cells. The aim of this study was to analyse the effect of the structural characteristics of biomimetic calcium deficient hydroxyapatite (CDHA) on the innate immune response of macrophages and the subsequent impact on osteogenesis and osteoclastogenesis. Murine RAW 264.7 cells were cultured, under standard and inflammatory conditions, on chemically identical CDHA substrates that varied in microstructure and porosity. The impact on osteogenesis was evaluated by incubating osteoblastic cells (SaOS-2) with RAW-CDHA conditioned extracts. The results showed that macrophages were sensitive to different textural and structural properties of CDHA. Under standard conditions, the impact of inflammatory cytokine production by RAW cells cultured on CDHA played a significant role in the degradation of substrates, suggesting the impact of resorptive behaviour of RAW cells on biomimetic surfaces. Osteoblast differentiation was stimulated by the conditioned media collected from RAW cells cultured on needle-like nanostructured CDHA. The results demonstrated that needle-like nanostructured CDHA was able to generate a favourable osteoimmune environment to regulate osteoblast differentiation and osteogenesis. Under inflammatory conditions, the incubation of RAW cells with less porous CDHA resulted in a decreased gene expression and release of pro-inflammatory cytokines.

JTD Keywords: Calcium phosphates, Biomimetic hydroxyapatite, Osteoimmunomodulation, Inflammation, Osteogenesis, Osteoclastogesis

Immune cells play a vital role in regulating bone dynamics. This has boosted the interest in developing biomaterials that can modulate both the immune and skeletal systems. In this study, calcium phosphates discs (i.e., beta-tricalcium phosphate, β-TCP) are functionalized with heparin to investigate the effects on immune and stem cell responses. The results show that the functionalized surfaces downregulate the release of hydrogen peroxide and proinflammatory cytokines (tumor necrosis factor alpha and interleukin 1 beta) from human monocytes and neutrophils, compared to nonfunctionalized discs. The macrophages show both elongated and round shapes on the two ceramic substrates, but the morphology of cells on heparinized β-TCP tends toward a higher elongation after 72 h. The heparinized substrates support rat mesenchymal stem cell (MSC) adhesion and proliferation, and anticipate the differentiation toward the osteoblastic lineage as compared to β-TCP and control. The coupling between the inflammatory response and osteogenesis is assessed by culturing MSCs with the macrophage supernatants. The downregulation of inflammation in contact with the heparinized substrates induces higher expression of bone-related markers by MSCs.

JTD Keywords: Calcium phosphates, Heparinization, Inflammation, Osteogenesis

Resveratrol and gallic acid, a lipophilic and a hydrophilic phenol, were co-loaded in innovative, biocompatible nanovesicles conceived for ensuring the protection of the skin from oxidative- and inflammatory-related affections. The basic vesicles, liposomes and glycerosomes, were produced by a simple, one-step method involving the dispersion of phospholipid and phenols in water or water/glycerol blend, respectively. Liposomes and glycerosomes were modified by the addition of poloxamer, a stabilizer and viscosity enhancer, thus obtaining viscous or semisolid dispersions of structured vesicles. The vesicles were spherical, unilamellar and small in size (~70 nm in diameter). The superior ability of the poloxamer-structured vesicles to promote the accumulation of both phenols in the skin was demonstrated, as well as their low toxicity and great ability to protect fibroblasts from chemically-induced oxidative damage. The in vivo administration of the vesicular phenols on TPA (phorbol ester)-exposed skin led to a significant reduction of oedema and leukocyte infiltration.

JTD Keywords: Fibroblasts, Mice, Phenol, Phospholipid vesicle, Poloxamer, Skin inflammation

Objectives: The goal of this study was to assess mitochondrial function, energy, and purine metabolism, protein synthesis machinery from the nucleolus to the ribosome, inflammation, and expression of newly identified ectopic olfactory receptors (ORs) and taste receptors (TASRs) in the frontal cortex of typical cases of dementia with Lewy bodies (DLB) and cases with rapid clinical course (rpDLB: 2 years or less) compared with middle-aged non-affected individuals, in order to learn about the biochemical abnormalities underlying Lewy body pathology. Methods: Real-time quantitative PCR, mitochondrial enzymatic assays, and analysis of β-amyloid, tau, and synuclein species were used. Results: The main alterations in DLB and rpDLB, which are more marked in the rapidly progressive forms, include (i) deregulated expression of several mRNAs and proteins of mitochondrial subunits, and reduced activity of complexes I, II, III, and IV of the mitochondrial respiratory chain; (ii) reduced expression of selected molecules involved in energy metabolism and increased expression of enzymes involved in purine metabolism; (iii) abnormal expression of nucleolar proteins, rRNA18S, genes encoding ribosomal proteins, and initiation factors of the transcription at the ribosome; (iv) discrete inflammation; and (v) marked deregulation of brain ORs and TASRs, respectively. Severe mitochondrial dysfunction involving activity of four complexes, minimal inflammatory responses, and dramatic altered expression of ORs and TASRs discriminate DLB from Alzheimer’s disease. Altered solubility and aggregation of α-synuclein, increased β-amyloid bound to membranes, and absence of soluble tau oligomers are common in DLB and rpDLB. Low levels of soluble β-amyloid are found in DLB. However, increased soluble β-amyloid 1–40 and β-amyloid 1–42, and increased TNFα mRNA and protein expression, distinguish rpDLB. Conclusion: Molecular alterations in frontal cortex in DLB involve key biochemical pathways such as mitochondria and energy metabolism, protein synthesis, purine metabolism, among others and are accompanied by discrete innate inflammatory response.

JTD Keywords: Dementia with Lewy bodies, Alzheimer’s disease, α-synuclein, Mitochondria, Protein synthesis, Inflammation, β-amyloid, Olfactory receptors

In the present work new highly biocompatible nanovesicles were developed using polyanion sodium hyaluronate to form polymer immobilized vesicles, so called hyalurosomes. Curcumin, at high concentration was loaded into hyalurosomes and physico-chemical properties and in vitro/in vivo performances of the formulations were compared to those of liposomes having the same lipid and drug content. Vesicles were prepared by direct addition of dispersion containing the polysaccharide sodium hyaluronate and the polyphenol curcumin to a commercial mixture of soy phospholipids, thus avoiding the use of organic solvents. An extensive study was carried out on the physico-chemical features and properties of curcumin-loaded hyalurosomes and liposomes. Cryogenic transmission electron microscopy and small-angle X-ray scattering showed that vesicles were spherical, uni- or oligolamellar and small in size (112-220 nm). The in vitro percutaneous curcumin delivery studies on intact skin showed an improved ability of hyalurosomes to favour a fast drug deposition in the whole skin. Hyalurosomes as well as liposomes were biocompatible, protected in vitro human keratinocytes from oxidative stress damages and promoted tissue remodelling through cellular proliferation and migration. Moreover, in vivo tests underlined a good effectiveness of curcumin-loaded hyalurosomes to counteract 12-O-tetradecanoilphorbol (TPA)-produced inflammation and injuries, diminishing oedema formation, myeloperoxydase activity and providing an extensive skin reepithelization. Thanks to the one-step and environmentally-friendly preparation method, component biocompatibility and safety, good in vitro and in vivo performances, the hyalurosomes appear as promising nanocarriers for cosmetic and pharmaceutical applications.

JTD Keywords: Cell oxidative stress, Hyaluronic acid/Hyaluronan, Phospholipid vesicles, Polyphenols, Skin inflammation, Wound healing

Bone marrow-derived mesenchymal stem cells (MSCs) reduce acute lung injury in animals challenged by bleomycin or bacterial lipopolysaccaride. It is not known, however, whether MSCs protect from ventilator-induced lung injury (VILI). This study investigated whether MSCs have a potential role in preventing or modulating VILI in healthy rats subjected to high-volume ventilation. 24 Sprague-Dawley rats (250-300 g) were subjected to high-volume mechanical ventilation (25 mL.kg(-1)). MSCs (5 x 10(6)) were intravenously or intratracheally administered (n=8 each) 30 min before starting over-ventilation and eight rats were MSC-untreated. Spontaneously breathing anesthetised rats (n=8) served as controls. After 3 h of over-ventilation or control the animals were sacrificed and lung tissue and bronchoalveolar lavage fluid (BALF) were sampled for further analysis. When compared with controls, MSC-untreated over-ventilated rats exhibited typical VILI features. Lung oedema, histological lung injury index, concentrations of total protein, interleukin-1 beta, macrophage inflammatory protein-2 and number of neutrophils in BALF and vascular cell adhesion protein-1 in lung tissue significantly increased in over-ventilated rats. All these indices of VILI moved significantly towards normalisation in the rats treated with MSCs, whether intravenously or intratracheally. Both local and systemic pre-treatment with MSCs reduced VILI in a rat model.

JTD Keywords: Acute lung injury, Cell therapy, Injurious ventilation, Lung inflammation, Lung oedema, Mechanical ventilation

Controlling the fate of implanted hMSCs is one of the major drawbacks to be overcome to realize tissue engineering strategies. In particular, the effect of the inflammatory environment on hMSCs behaviour is poorly understood. Studying and mimicking the inflammatory process in vitro is a very complex and challenging task that involves multiple variables. This research addressed the questions using in vitro co-cultures of primary derived hMSCs together with human peripheral blood mononucleated cells (PBMCs); the latter are key agents in the inflammatory process. This work explored the in vitro phenotypic changes of hMSCs in co-culture direct contact with monocytes and lymphocytes isolated from blood using both basal and osteogenic medium. Our findings indicated that hMSCs maintained their undifferentiated phenotype and pluripotency despite the contact with PBMCs. Moreover, hMSCs demonstrated increased proliferation and were able to differentiate specifically down the osteogenic lineage pathway. Providing significant crucial evidence to support the hypothesis that inflammation and host defence mechanisms could be utilised rather than avoided and combated to provide for the successful therapeutic application of stem cell therapies.

JTD Keywords: Co-culture, Inflammation, Mesenchymal stem cells, Monocytes, Osteoblasts

Background: Obstructive sleep apnea (OSA) is associated with cardiovascular disorders, but the different comorbidities in OSA patients make it difficult to know their specific effects on the development of cardiovascular injury. The aim of the present study was to investigate whether recurrent obstructive apneas could lead to myocardial injury. Methods: Thirty-six male Sprague-Dawley rats (300-350. g) were either acutely (3. h) or sustainably (5. h/day, for 10. days) subjected to obstructive apneas with a pattern of 15. s each, 60. apneas/h. Corresponding control groups were formed for the acute and sustained models. To assess the induction of systemic inflammation, IL1-β was measured in plasma. Ventricular tissue injury was evaluated by histological techniques (presence of inflammatory cell infiltration, eosin autofluorescence, and detection of apoptosis). Results: After 3. h of obstructive apneas, a significant increase in IL1-β (64.9. ±. 29.6. ng/μl) were observed with respect to the controls (7.3. ±. 1.0. ng/μl), but no myocardial injury was present. Conversely to the acute model, the systemic inflammation triggered by obstructive apneas for 10. days was reduced. However, the percentage of area with enhanced eosin autofluorescence and of apoptotic cells (1.83. ±. 0.35% and 24.4. ±. 1.5%, respectively) was increased when compared to the control group (0.72. ±. 0.20% and 5.0. ±. 2.8%, respectively). Conclusions: This study suggests that obstructive apneas are a potential source of early systemic and ventricular inflammation and myocardial cell injury after sustained apneas application, which could represent an initial phase in the progression of heart disease associated with OSA.

JTD Keywords: Animal models, Inflammation, Myocardial injury, Obstructive sleep apnea

Cognitive impairment is one of the main consequences of obstructive sleep apnea (OSA) and is usually attributed in part to the oxidative stress caused by intermittent hypoxia in cerebral tissues. The presence of oxygen-reactive species in the brain tissue should be produced by the deoxygenation-reoxygenation cycles which occur at tissue level during recurrent apneic events. However, how changes in arterial blood oxygen saturation (SpO(2)) during repetitive apneas translate into oxygen partial pressure (PtO2) in brain tissue has not been studied. The objective of this study was to assess whether brain tissue is partially protected from intermittently occurring interruption of O-2 supply during recurrent swings in arterial SpO(2) in an animal model of OSA. Methods: Twenty-four male Sprague-Dawley rats (300-350 g) were used. Sixteen rats were anesthetized and noninvasively subjected to recurrent obstructive apneas: 60 apneas/h, 15 s each, for 1 h. A control group of 8 rats was instrumented but not subjected to obstructive apneas. PtO2 in the cerebral cortex was measured using a fast-response oxygen microelectrode. SpO(2) was measured by pulse oximetry. The time dependence of arterial SpO(2) and brain tissue PtO2 was carried out by Friedman repeated measures ANOVA. Results: Arterial SpO(2) showed a stable periodic pattern (no significant changes in maximum [95.5 +/- 0.5%; m +/- SE] and minimum values [83.9 +/- 1.3%]). By contrast, brain tissue PtO2 exhibited a different pattern from that of arterial SpO(2). The minimum cerebral cortex PtO2 computed during the first apnea (29.6 +/- 2.4 mmHg) was significantly lower than baseline PtO2 (39.7 +/- 2.9 mmHg; p = 0.011). In contrast to SpO(2), the minimum and maximum values of PtO2 gradually increased (p < 0.001) over the course of the 60 min studied. After 60 min, the maximum (51.9 +/- 3.9 mmHg) and minimum (43.7 +/- 3.8 mmHg) values of PtO2 were significantly greater relative to baseline and the first apnea dip, respectively. Conclusions: These data suggest that the cerebral cortex is partially protected from intermittently occurring interruption of O-2 supply induced by obstructive apneas mimicking OSA.

JTD Keywords: Near-infrared spectroscopy, Sleep-apnea, Iintermittent hypoxia, Cerebral oxygenation, Oxidative stress, Blood-flow, Rat, Apoptosis, Inflammation, Hypercapnia

Background: The aim was to test the hypothesis that the blood serum of rats subjected to recurrent airway obstructions mimicking obstructive sleep apnea (OSA) induces early activation of bone marrow-derived mesenchymal stem cells (MSC) and enhancement of endothelial wound healing. Methods: We studied 30 control rats and 30 rats subjected to recurrent obstructive apneas (60 per hour, lasting 15 s each, for 5 h). The migration induced in MSC by apneic serum was measured by transwell assays. MSC-endothelial adhesion induced by apneic serum was assessed by incubating fluorescent-labelled MSC on monolayers of cultured endothelial cells from rat aorta. A wound healing assay was used to investigate the effect of apneic serum on endothelial repair. Results: Apneic serum showed significant increase in chemotaxis in MSC when compared with control serum: the normalized chemotaxis indices were 2.20 +/- 0.58 (m +/- SE) and 1.00 +/- 0.26, respectively (p < 0.05). MSC adhesion to endothelial cells was greater (1.75 +/- 0.14 -fold; p < 0.01) in apneic serum than in control serum. When compared with control serum, apneic serum significantly increased endothelial wound healing (2.01 +/- 0.24 -fold; p < 0.05). Conclusions: The early increases induced by recurrent obstructive apneas in MSC migration, adhesion and endothelial repair suggest that these mechanisms play a role in the physiological response to the challenges associated to OSA.

JTD Keywords: Induced acute lung, Sleep-apnea, Intermitent hypoxia, Cardiovascular-disease, Progenito Cells, Rat model, Inflammation, Mechanisms, Repair, Blood

The aim was to test the hypothesis that mesenchymal stem cells (MSC) could reduce the inflammation induced by recurrent airway occlusions in an animal model of obstructive sleep apnea (OSA). A nasal mask was applied to 30 anesthetized rats. Twenty rats were subjected to a pattern of recurrent obstructive apneas mimicking OSA (60/h, lasting 15 s each) for 5h. MSC (5x10(6) cells) were intravenously injected into 10 of these rats. Ten rats not subjected to apneas or MSC injection were used as controls. The rat blood serum concentrations of pro-inflammatory cytokine IL-1beta were measured by ELISA. IL-1beta was significantly greater in the rats subjected to recurrent apneas (66.7+/-41.2 pg/mL; m+/-SEM) than in controls (1.9+/-1.0 pg/mL; p<0.05). In the group of apneic rats subjected to MSC injection, IL-1beta was significantly reduced (6.1+/-3.8 pg/mL; p<0.05). In conclusion, MSC triggered an early anti-inflammatory response in rats subjected to recurrent obstructive apneas, suggesting that these stem cells could play a role in the physiological response to counterbalance inflammation in OSA.

JTD Keywords: Obstructive sleep apnea, Animal model, Airway obstruction, Inflammation

The upper airway of obstructive sleep apnoea patients is subjected to recurrent negative pressure swings promoting its collapse and reopening. The aim of the present study was to ascertain whether this mechanical stress induces upper airway inflammation in a rat model. The upper airway of Sprague-Dawley rats was subjected to a periodic pattern of recurrent negative (-40 cmH2O, 1 s) and positive (4 cmH2O, 2 s) pressures inducing collapse and reopening for 5 h. Rats that were instrumented but not subjected to negative pressure swings were used as controls. The gene expression of the pro-inflammatory biomarkers macrophage inflammatory protein (MIP)-2, tumour necrosis factor (TNF)-alpha, interleukin (IL)-1beta and P-selectin in the soft palate and larynx tissues was assessed by real-time PCR. A marked overexpression of MIP-2, TNF-alpha, IL-1beta and P-selectin (approximately 40-, 24-, 47- and 7-fold greater than controls, respectively) was observed in the larynx tissue; similar results were found in the soft palate tissue (approximately 14-, 7-, 35- and 11-fold greater than controls, respectively). Recurrent upper airway collapse and reopening mimicking those experienced by obstructive sleep apnoea patients triggered an early local inflammatory process. These results could explain the inflammation observed in the upper airway of obstructive sleep apnoea patients.

JTD Keywords: Airway collapse, Airway reopening, Inflammation, Negative pressure, Obstructive sleep apnoea, Upper airway