Aging hampers neutrophil extracellular traps (NETs) efficacy.

BACKGROUND: NETosis is a neutrophil-mediated defense mechanism during which DNA and enzymes are extruded forming a network (NETs) trapping and killing different pathogens. NETosis is reduced in both mice and humans during aging. AIMS: We explored the difference in the efficacy of NETs released in elderly (> 65 years) versus adults (20-50 years) subjects in inhibiting Staphylococcus aureus growth and activating the growth of keratinocytes. METHODS: Neutrophil granulocytes, obtained from venous blood both in healthy elderly and adult subjects, were stimulated by LPS (0-250 µg/ml) to induce the formation of NET. NETs were quantified by SYBR Green staining and growth inhibition of S. aureus was evaluated by disk diffusion test. Furthermore, NETs (0-500 ng/ml) were added to immortalized human keratinocytes (HaCaT cells), and their proliferation was evaluated by MTT assay after 24 h. Finally, the DNA size of NETs was evaluated by flow cytometry after SYBR Green staining. RESULTS: Greater production of NETs was observed in elderly subjects than in adults, but these NETs showed reduced bactericidal capacity and HaCaT cells' proliferation stimulation. The activities of the NETs are related to the size of the extruded DNA threads, and when NETs size was analyzed, DNA from elderly showed a higher size compared to that obtained by adults. DISCUSSION: Unexpected results showed aging-related NETs structural modification resulting in both a lower antimicrobial activity and keratinocyte proliferation stimulation compared to NETs obtained from adults. CONCLUSIONS: The NETs DNA size observed in elderly subjects has not been previously reported and could be part of other pathogenic mechanisms observed in aging.

3D Bioprinting of Gelatin-Xanthan Gum Composite Hydrogels for Growth of Human Skin Cells.

In recent years, bioprinting has attracted much attention as a potential tool for generating complex 3D biological constructs capable of mimicking the native tissue microenvironment and promoting physiologically relevant cell-cell and cell-matrix interactions. The aim of the present study was to develop a crosslinked 3D printable hydrogel based on biocompatible natural polymers, gelatin and xanthan gum at different percentages to be used both as a scaffold for cell growth and as a wound dressing. The CellInk Inkredible 3D printer was used for the 3D printing of hydrogels, and a glutaraldehyde solution was tested for the crosslinking process. We were able to obtain two kinds of printable hydrogels with different porosity, swelling and degradation time. Subsequently, the printed hydrogels were characterized from the point of view of biocompatibility. Our results showed that gelatin/xanthan-gum bioprinted hydrogels were biocompatible materials, as they allowed both human keratinocyte and fibroblast in vitro growth for 14 days. These two bioprintable hydrogels could be also used as a helpful dressing material.

Verteporfin-Loaded Mesoporous Silica Nanoparticles' Topical Applications Inhibit Mouse Melanoma Lymphangiogenesis and Micrometastasis In Vivo.

Photodynamic therapy (PDT) has been pointed out as a candidate for improving melanoma treatment. Nanotechnology application in PDT has increased its efficacy by reducing side effects. Herein, mesoporous silica nanoparticles (MSNs) conjugated with verteporfin (Ver-MSNs), in use with PDT, were administered in mice to evaluate their efficacy on lymphoangiogenesis and micrometastasis in melanoma. Melanoma was induced in mice by the subcutaneous injection of B16-F10 cells. The mice were transcutaneously treated with MSNs, Ver-MSNs, or glycerol and exposed to red light. The treatment was carried out four times until day 20. Lymphangiogenesis and micrometastasis were identified by the immunohistochemical method. Lymphoangiogenesis was halved by MSN treatment compared with the control animals, whereas the Ver-MSN treatment almost abolished it. A similar reduction was also observed in lung micrometastasis. PDT with topically administrated Ver-MSNs reduced melanoma lymphoangiogenesis and lung micrometastasis, as well as tumor mass and angiogenesis, and therefore their use could be an innovative and useful tool in melanoma clinical therapy.

Gastric distension causes changes in heart rate and arterial blood pressure by affecting the crosstalk between vagal and splanchnic systems in anesthetised rats.

Various hindbrain nuclei have been demonstrated to be involved in the control of the cardiovascular reflexes elicited by both non-noxious and noxious gastric distension, through parasympathetic and sympathetic activation. The different role played by the branches of autonomic nervous system in exerting these effects and their crosstalk in relation to low-/high-pressure distension rate has not been examined yet. Therefore, in the present work, monolateral and bilateral vagotomy and splanchnicotomy were performed in anesthetised rats to analyse the involvement of hindbrain nuclei in haemodynamic changes caused by gastric distension at high (80 mmHg) and low (15 mmHg) pressure. The analysis of c-Fos expression in neuronal areas involved in cardiovascular control allowed us to examine their recruitment in response to various patterns of gastric distension and the crosstalk between vagal and splanchnic systems. The results obtained show that the low-pressure (non-noxious) gastric distension increases both heart rate and arterial blood pressure. In addition, the vagus nerve and hindbrain nuclei, such as nucleus ambiguous, ventrolateral medulla and lateral reticular nucleus, appear to be primarily involved in observed responses. In particular, we have found that although vagus nerve plays a central role in exerting those cardiovascular reflex changes at low gastric distension, for its functional expression an intact splanchnic system is mandatory. Hence, the absence of splanchnic input attenuates pressor responses or turns them into depressor responses. Instead at high-pressure (noxious) gastric distension, the splanchnic nerve represents the primary component in regulating the reflex cardiovascular effects.

Effect of retinoic acid and vitamin D3 on osteoblast differentiation and activity in aging.

Several studies have evidenced that in aging, osteoblast functional activity is impaired: osteoblast proliferation is slower and matrix deposition is less efficient. Because peroxisome-proliferator-activated receptor γ2 (PPARγ2) and fatty acids are important inhibitory signals in osteoblast development, we have investigated in human primary osteoblasts obtained from patients of different ages, the influence of retinoic acid and calcitriol on enzymes involved in differentiative (PPARγ2, ß-catenin, and insulin-like growth factor 1) and metabolic (carnitine palmitoyltransferase 1) intracellular pathways, and on transglutaminase 2, as enzyme fundamental for stabilizing the newly deposited extracellular matrix in bone. Retinoic acid and calcitriol influenced, respectively, proliferation and differentiation of osteoblasts, and an increase in PPARγ2 expression was observed following retinoic acid administration, whereas a decrease was observed following calcitriol administration. Aging widely influenced all parameters analyzed (the proliferation, differentiation, and new matrix deposition are significantly reduced in aged osteoblasts), with the exception of PPARγ2, which we found to be constitutively overexpressed and not modulated by retinoic acid or calcitriol administration. Our findings show the impaired ability of aged osteoblasts to perform adequate functional response and draw attention to the therapeutic approaches for bone healing in elderly patients.

A study of the mechanical properties of ePTFE suture used as artificial mitral chordae.

BACKGROUND AND AIM OF THE STUDY: We investigated the dimensional and mechanical properties of polyetetrafluorene (ePTFE) sutures used as artificial chordae during mitral valve repair. METHODS: Mechanical properties of ePTFE synthetic chordae tendineae were tested with a servo hydraulic testing machine. Several different lengths from 2 to 14 cm were studied under both single and multiple mechanical traction. RESULTS: The mechanical behavior of artificial chordae reveals that three centimeters is the length over which we observe a significant increase in stiffness. The chordae stiffness grows further at the length greater than seven centimeters following a low number of traction cycles. CONCLUSION: The increase of the length of artificial ePTFE chordae is accompanied by an increasing stiffness that compromises the long-term resistance of the chordae. ePTFE length can alter the performance of artificial chordae. This suggests that mitral valve repairs which anchor ePTFE neochordae to the ventricular apex may have less durability than when anchored to the tips of the papillary muscles.

Polydopamine Blending Increases Human Cell Proliferation in Gelatin-Xanthan Gum 3D-Printed Hydrogel.

BACKGROUND: Gelatin-xanthan gum (Gel-Xnt) hydrogel has been previously modified to improve its printability; now, to increase its ability for use as cell-laden 3D scaffolds (bioink), polydopamine (PDA), a biocompatible, antibacterial, adhesive, and antioxidant mussel-inspired biopolymer, has been added (1-3% v/v) to hydrogel. METHODS: Control (CT) and PDA-blended hydrogels were used to print 1 cm2 grids. The hydrogels' printability, moisture, swelling, hydrolysis, and porosity were tested after glutaraldehyde (GTA) crosslinking, while biocompatibility was tested using primary human-derived skin fibroblasts and spontaneously immortalized human keratinocytes (HaCaT). Keratinocyte or fibroblast suspension (100 µL, 2.5 × 105 cells) was combined with an uncrosslinked CT and PDA blended hydrogel to fabricate cylinders (0.5 cm high, 1 cm wide). These cylinders were then cross-linked and incubated for 1, 3, 7, 14, and 21 days. The presence of cells within various hydrogels was assessed using optical microscopy. RESULTS AND DISCUSSION: PDA blending did not modify the hydrogel printability or physiochemical characteristics, suggesting that PDA did not interfere with GTA crosslinking. On the other hand, PDA presence strongly accelerated and increased both fibroblast and keratinocyte growth inside. This effect seemed to be linked to the adhesive abilities of PDA, which improve cell adhesion and, in turn, proliferation. CONCLUSIONS: The simple PDA blending method described could help in obtaining a new bioink for the development of innovative 3D-printed wound dressings.

Epiregulin induces human SK-N-BE cell differentiation through ERK1/2 signaling pathway.

Epidermal growth factor (EGF) and other EGF-related growth factors, such as transforming growth factor-α, are able to stimulate neuroblastoma (NB) cell proliferation. Epiregulin (Epi) is a growth factor belonging to the EGF family known to be more potent than EGF in mediating mitogenic signals. In this study, we tested the ability of Epi to stimulate a human NB cell line (SK-N-BE) proliferation. Surprisingly, Epi (50-1000 ng/ml) induced a reduction in SK-N-BE proliferation along with a morphological differentiation, associated with an increase in MMP-9 expression. Moreover, Epi-induced differentiation was inhibited by ERK1/2 phosphorilation inhibition. In conclusion, Epi could represent a novel and useful tool to oppose NB cell proliferation.

Effects and differentiation activity of IGF-I, IGF-II, insulin and preptin on human primary bone cells.

The importance of the complex interrelated regulatory pathways involving IGF factors and pancreatic hormones can be observed in several metabolic diseases, where the deregulation of these factors has a wide impact on bone health. These findings have stimulated us to compare the effect of IGF-I, IGF-II, insulin and preptin on human bone cells. The effect on cell differentiation and cell activity of osteoblasts and osteoclasts has been analysed. We have observed a significant effect by IGF-I, a modest effect by IGF-II and preptin and no effect after insulin administration on human primary osteoblast-like cells. All studied factors have shown an induction on human primary osteoclast differentiation and bone resorption activity, with IGF-I being the most potent factor. We hypothesize that these findings may be on the basis of decreased bone mass density observed in several diseases.

Breaking a Vicious Circle: Lymphangiogenesis as a New Therapeutic Target in Wound Healing.

The lymphatic system is of fundamental importance in maintaining a fluid balance in the body and tissue homeostasis; it drains protein-rich lymph from the interstitial space and facilitates the release of cells that mediate the immune response. When one tissue is damaged, more cells and tissues work to repair the damaged site. Blood and lymph vessels are particularly important for tissue regeneration and healing. Angiogenesis is the process of the formation of new blood vessels and is induced by angiogenic factors such as VEGF-A; VEGF-C/D-induced lymphangiogenesis and both occur simultaneously during wound healing. After the inflammatory phase, lymphatic vessels suppress inflammation by aiding in the drainage of inflammatory mediators; thus, disorders of the lymphatic system often result in chronic and disabling conditions. It has recently been clarified that delayed wound healing, as in diabetes, can occur as a consequence of impaired lymphangiogenesis. In this review, we have highlighted recent advances in understanding the biology underlying lymphangiogenesis and its key role in wound healing, and the possibility of its pharmacological modulation as a novel therapeutic strategy for the treatment of chronic wounds.

RETRACTION: Intrinsic innervation and dopaminergic markers after experimental denervation in rat thymus.

On behalf of the coauthors and with much regret, I must retract our publication entitled "Intrinsic innervation and dopaminergic markers after experimental denervation in rat thymus" published in European Journal of Histochemistry 2010;54(2):e17 for the following reason: Unfortunately, now, after thirteen years, we have realized that some microphotographs published in the paper have been processed to improve the presentation of the images. The three surviving authors of the paper agree that the processing of the presentation images is against the COPE Ethical Editorial Standard, although the presentation images do not alter the integrity of methodological procedures and the results of the research work, obtained from the direct analysis of slides under microscope and rigorous statistical analysis of data; therefore, we, the authors of the above indicated paper, request the retraction of the publication. We apologize for what happened.   Maurizio Sabbatini Dip. di Scienze e Innovazione Tecnologica (DISIT) Università del Piemonte Orientale Alessandria, Italy.

Laser Biostimulation Induces Wound Healing-Promoter ß2-Defensin Expression in Human Keratinocytes via Oxidative Stress.

The innate immune system is the first line of defense of the body composed of anatomical barriers, such as skin and mucosa, as well as effector cells, antimicrobial peptides, soluble mediators, and cell receptors able to detect and destroy viruses and bacteria and to sense trauma and wounds to initiate repair. The human ß-defensins belong to a family of antimicrobial small cationic peptides produced by epithelial cells, and show immunomodulatory and pro-healing activities. Laser biostimulation is a therapy widely used to contrast microbial infection and to accelerate wound healing through biological mechanisms that include the creation of oxidative stress. In this paper, we explored laser biostimulation's ability to modulate the production of two ß-defensins, hBD-1 and hBD-2, in human keratinocytes and whether this modulation was, at least in part, oxidative-stress-dependent. Human spontaneously immortalized keratinocytes (HaCaT) were stimulated using laser irradiation at a 980 nm wavelength, setting the power output to 1 W (649.35 mW/cm2) in the continuous mode. Cells were irradiated for 0 (negative control), 5, 10, 25 and 50 s, corresponding to an energy stimulation of 0, 5, 10, 25 and 50 J. Positive control cells were treated with lipopolysaccharide (LPS, 200 ng/mL). After 6 and 24 h of treatment, the cell conditioned medium was collected and analyzed via ELISA assay for the production of hBD-1 and hBD-2. In another set of experiments, HaCaT were pre-incubated for 45 min with antioxidant drugs-vitamin C (Vit. C, 100 µM), sodium azide (NaN3, 1 mM); ω-nitro-L-arginine methyl ester (L-NAME, 10 mM) and sodium pyruvate (NaPyr, 100 µM)-and then biostimulated for 0 or 50 s. After 6 h, the conditioned medium was collected and used for the ELISA analysis. The hBD-1 and hBD-2 production by HaCaT was significantly increased by single laser biostimulation after 6 h in an energy-dependent fashion compared to basal levels, and both reached production levels induced by LPS. After 24 h, only hBD-2 production induced by laser biostimulation was further increased, while the basal and stimulated hBD-1 levels were comparable. Pre-incubation with antioxidative drugs was able to completely abrogate the laser-induced production of both hBD-1 and hBD-2 after 6 h, with the exception of hBD-1 production in samples stimulated after NaN3 pre-incubation. A single laser biostimulation induced the oxidative-stress-dependent production of both hBD-1 and hBD-2 in human keratinocytes. In particular, the pro-healing hBD-2 level was almost three times higher than the baseline level and lasted for 24 h. These findings increase our knowledge about the positive effects of laser biostimulation on wound healing.

The importance of gravity vector on adult mammalian organisms: Effects of hypergravity on mouse testis.

In the age of space exploration, the effect of hypergravity on human physiology is a relatively neglected topic. However, astronauts have several experiences of hypergravity during their missions. The main disturbance of altered gravity can be imputed to cell cytoskeleton alteration and physiologic homeostasis of the body. Testis has proved to be a particularly sensible organ, subject to environmental alteration and physiological disturbance. This makes testis an organ eligible for investigating the alteration following exposure to altered gravity. In our study, mice were exposed to hypergravity (3g for 14 days) in the Large Diameter Centrifuge machine (ESA, Netherland). We have observed a morphological alteration of the regular architecture of the seminiferous tubules of testis as well as an altered expression of factors involved in the junctional complexes of Sertoli cells, responsible for ensuring the morpho-functional integrity of the organ. The expression of key receptors in physiological performance, such as Androgen Receptors and Interstitial Cells Stimulating Hormone receptors, was found lower expressed. All these findings indicate the occurrence of altered physiological organ performance such as the reduction of the spermatozoa number and altered endocrine parameters following hypergravity exposure.

Delirium risk factors analysis post proximal femur fracture surgery in elderly.

BACKGROUND AND AIM: The increase in the average-age and in the percentage of elderly people implies an exponential increase in fractures of the proximal femur. A common consequence of hip fracture in elderly patients is delirium, characterized by cognitive confusion or a lethargic-type condition. Predisposing factors have been identified, but risk factors assessment useful for managing clinical intervention, has not received unanimous consent. This work aims to identify the potential risk factors for delirium in the elderly operated for hip fracture. METHODS: In this prospective observational study, we included 83 patients aged ≥65 years. Patients undergoing osteosynthesis of the femur and hip replacement for fractures were included. Patients already delusional in the pre-operative period were excluded. At the time, deadlines T0 (pre-operative), and T1,T3,T7 post-operative day, delirium, hematic parameters, blood transfusions, were assessed. RESULTS: Level of delirium was assessed obtaining 80% not delusional and 20% delusional. Glycemia and hemoglobin were not found to be risk factors, although they are known to influence cognitive status; we hypothesize they should be considered predisposing factors. Comorbidities such as atrial fibrillation and Chronic Obstructive Pulmonary Disease were found associated with delirium. The most advanced age, anxiolytic drugs, the use of benzodiazepine as anaesthetic, the time surgical waiting, were found significantly associated with delirium. CONCLUSIONS: Taken together, findings of this prospective observational study showed that environmental and metabolic risk factors might contribute to make elderly susceptible to develop postoperative delirium following hip surgery. Thus, these patients should be adequately assessed and monitored. (www.actabiomedica.it).

NETosis in Wound Healing: When Enough Is Enough.

The neutrophils extracellular traps (NETs) are a meshwork of chromatin, histonic and non-histonic proteins, and microbicidal agents spread outside the cell by a series of nuclear and cytoplasmic events, collectively called NETosis. NETosis, initially only considered a defensive/apoptotic mechanism, is now considered an extreme defensive solution, which in particular situations induces strong negative effects on tissue physiology, causing or exacerbating pathologies as recently shown in NETs-mediated organ damage in COVID-19 patients. The positive effects of NETs on wound healing have been linked to their antimicrobial activity, while the negative effects appear to be more common in a plethora of pathological conditions (such as diabetes) and linked to a NETosis upregulation. Recent evidence suggests there are other positive physiological NETs effects on wound healing that are worthy of a broader research effort.

Processing Adipose Tissue to Make it More Stable When Used for Refilling: A Morphologic and Immunohistochemistry Evaluation.

Breast reconstruction has gained from lipofilling the possibility to recover the aesthetic outcome of anatomical profile in a more natural appearance. However, until today, the long-term graft survival remains unpredictable, and sometimes it does not guarantee a well-adequate aesthetic result. In the present work, the morphological changes, occurring in fat mass used for refilling, harvested by the Coleman's procedure or through the washing/fragmenting procedure were analysed. Adipocyte size; immunohistochemistry against CD8, CD31, CD68 and M2-type macrophages and catalase enzyme, were analysed in vitro on fat mass cultured for 4 weeks. Our observation reveals an increase of connective tissue around the mass and a high number of immune cells occurrence in fat mass harvested by the Coleman's procedure. Instead, the washing/fragmented procedure would reduce the number of immune cells within the fat mass, increase the size of adipocytes, and cause a wider presence of active vessels profile and greater catalase expression. We hypothesize that the fat mass processed by the Coleman's procedure would remain more reactive due to a higher number of immune and macrophages cells, prone to develop cystic formation, leading to a suboptimal integration in the recipient site. On the other hand, the conditions more prone to realize an optimal integration would occur in the fat mass processed by the washing/fragmenting procedure: a reduced number of immune cells, low amount of connective tissue, presence of larger adipocytes. Follow-up monitoring did support our conclusion, as we observed a reduction of re-intervention for refilling procedure in patients treated with the washing/fragmenting procedure.

Dopamine receptor subtypes in the native human heart.

The present study first time detected D1-D5 dopamine receptor subtypes in the native human heart simultaneously, found presence of D1, D2, D4, and D5 in cardiac tissues, and revealed distribution features of dopamine receptor subtypes in the epicardium, myocardium, and endocardium. Samples from four native hearts coming from young brain-dead donors, which for technical reason were not used for transplants, were studied. Dopamine receptors were revealed by immunochemistry technique and immunoblot analysis. Morphometrical quantification of the density of each receptor subtype was performed by an image analyzer. Our results demonstrate that only four subtypes of dopamine receptors can be found in cardiac tissues: D1, D2, D4, and D5. These dopamine receptors have been detected in endocardium, myocardium, and epicardium. D1 receptors were stored primarily in the epicardial layer. Dopamine receptors are distributed in the wall of both atria and ventricles, and its transmural gradient can be described in the wall of the human heart. Sections of atria and ventricles exposed to antidopamine receptor antibodies showed fluorescent-positive reaction in the epicardium, myocardium and endocardium. D4 receptor immune reactivity was remarkably less intense than D2 receptor immune reactivity. All the subtypes of dopamine receptors are in close relationships with all cardiac structures. Our findings provide a favorable basis for researching the role of dopamine receptors in controlling functions of the human heart and in the pathogenesis of cardiovascular diseases.

Translation, cross-cultural adaptation, reliability, and validation of the italian version of the Foot and Ankle Disability Index (FADI).

BACKGROUND AND AIM OF THE WORK: Foot-and-Ankle-Disability-Index (FADI) is one of the most widely used evaluation questionnaires for this anatomical district, but an italian validated version lacks and is necessary to properly evaluate italian people. In fact a correct interpretation of the items by patients is essential to obtain a precise subjective response, making the questionnaire valid to evaluate patients' satisfaction and wellness. Our purpose was to translate and culturally adapt into Italian the FADI questionnaire, and to check its reproducibility and validity. MATERIALS AND METHODS: The original english version of FADI questionnaire was translated into Italian and checked for medical part coherence. It was submitted to 10 italian randomized patients to verify a correct cultural adaptation, and then to other 50 randomized patients operated at their ankle or hallux to assess intra- and inter-observer reproducibility by the Pearson's-Correlation-Coefficient (PCC) and the Intra-Class-Correlation (ICC) coefficient. Moreover, Short-Form-36 (SF36) questionnaire for Quality-of-Life and Visual-Analogue-Scale (VAS) for pain were also administered to the same 60 people and compared to italian-FADI to perform validation analysis by PCC and ICC coefficient. RESULTS: Cultural adaptation of the translated version of the scale resulted good in terms of understandability by patients. An optimal correlation of the inter- and intra-observer reproducibility was obtained. The correlation obtained between FADI and SF-36 as well as between FADI and VAS indicates success in the validation process. CONCLUSIONS: Validation of the FADI italian version has been performed successfully, its use can be considered appropriate and is indicated in italian clinical practice. (www.actabiomedica.it).

Pt(IV) Bifunctional Prodrug Containing 2-(2-Propynyl)octanoato Axial Ligand: Induction of Immunogenic Cell Death on Colon Cancer.

The synthesis, characterization, and in vitro activity of a cyclohexane-1 R,2 R-diamine-based Pt(IV) derivative containing the histone deacetylase inhibitor rac-2-(2-propynyl)octanoato, namely, ( OC-6-44)-acetatodichlorido(cyclohexane-1 R,2 R-diamine)( rac-2-(2-propynyl)octanoato)platinum(IV), are reported together with those of its isomers containing enantiomerically enriched axial ligands. These Pt(IV) complexes showed comparable activity, of 2 orders of magnitude higher than reference drug oxaliplatin on three human (HCT 116, SW480, and HT-29) and one mouse (CT26) colon cancer cell lines. In vivo experiments were carried out on immunocompetent BALB/c mice bearing the same syngeneic tumor. The complex ( OC-6-44)-acetatodichlorido(cyclohexane-1 R,2 R-diamine)( rac-2-(2-propynyl)octanoato)platinum(IV) showed higher tumor mass Pt accumulation than oxaliplatin, due to its higher lipophilicity, with negligible nephro- and hepatotoxicities when administered intravenously. A remarkable tumor mass invasion by cytotoxic CD8+ T lymphocytes, following the Pt(IV) treatment, indicated a strong induction of immunogenic cell death.