New orphan disease therapies from the proteome of industrial plasma processing waste- a treatment for aceruloplasminemia.

Plasma-derived therapeutic proteins are produced through an industrial fractionation process where proteins are purified from individual intermediates, some of which remain unused and are discarded. Relatively few plasma-derived proteins are exploited clinically, with most of available plasma being directed towards the manufacture of immunoglobulin and albumin. Although the plasma proteome provides opportunities to develop novel protein replacement therapies, particularly for rare diseases, the high cost of plasma together with small patient populations impact negatively on the development of plasma-derived orphan drugs. Enabling therapeutics development from unused plasma fractionation intermediates would therefore constitute a substantial innovation. To this objective, we characterized the proteome of unused plasma fractionation intermediates and prioritized proteins for their potential as new candidate therapies for human disease. We selected ceruloplasmin, a plasma ferroxidase, as a potential therapy for aceruloplasminemia, an adult-onset ultra-rare neurological disease caused by iron accumulation as a result of ceruloplasmin mutations. Intraperitoneally administered ceruloplasmin, purified from an unused plasma fractionation intermediate, was able to prevent neurological, hepatic and hematological phenotypes in ceruloplasmin-deficient mice. These data demonstrate the feasibility of transforming industrial waste plasma fraction into a raw material for manufacturing of new candidate proteins for replacement therapies, optimizing plasma use and reducing waste generation.

Covalently Binding Adenosine A3 Receptor Agonist ICBM Irreversibly Reduces Voltage-Gated Ca2+ Currents in Dorsal Root Ganglion Neurons.

Interest has been focused in recent years on the analgesic effects exerted by adenosine and its receptors, A1, A2A, A2B, and A3 adenosine receptor (AR) subtypes, in different in vivo models of chronic pain. In particular, it was demonstrated that selective A3AR agonists reduced pro-nociceptive N-type Ca2+ channels in dorsal root ganglion (DRG) neurons isolated from rats and, by this mechanism, inhibit post inflammatory visceral hypersensitivity. In the present study, we investigate the effect of a previously reported irreversibly binding A3AR agonist, ICBM, on Ca2+ currents (ICa) in rat DRG neurons. Present data demonstrate that ICBM, an isothiocyanate derivative designed for covalent binding to the receptor, concentration-dependently inhibits ICa. This effect is irreversible, since it persists after drug removal, differently from the prototypical A3AR agonist, Cl-IB-MECA. ICBM pre-exposure inhibits the effect of a subsequent Cl-IB-MECA application. Thus, covalent A3AR agonists such as ICBM may represent an innovative, beneficial, and longer-lasting strategy to achieve efficacious chronic pain control versus commonly used, reversible, A3AR agonists. However, the possible limitations of this drug and other covalent drugs may be, for example, a characteristic adverse effect profile, suggesting that more pre-clinical studies are needed.

Phenomic Microglia Diversity as a Druggable Target in the Hippocampus in Neurodegenerative Diseases.

Phenomics, the complexity of microglia phenotypes and their related functions compels the continuous study of microglia in disease animal models to find druggable targets for neurodegenerative disorders. Activation of microglia was long considered detrimental for neuron survival, but more recently it has become apparent that the real scenario of microglia morphofunctional diversity is far more complex. In this review, we discuss the recent literature on the alterations in microglia phenomics in the hippocampus of animal models of normal brain aging, acute neuroinflammation, ischemia, and neurodegenerative disorders, such as AD. Microglia undergo phenomic changes consisting of transcriptional, functional, and morphological changes that transform them into cells with different properties and functions. The classical subdivision of microglia into M1 and M2, two different, all-or-nothing states is too simplistic, and does not correspond to the variety of phenotypes recently discovered in the brain. We will discuss the phenomic modifications of microglia focusing not only on the differences in microglia reactivity in the diverse models of neurodegenerative disorders, but also among different areas of the brain. For instance, in contiguous and highly interconnected regions of the rat hippocampus, microglia show a differential, finely regulated, and region-specific reactivity, demonstrating that microglia responses are not uniform, but vary significantly from area to area in response to insults. It is of great interest to verify whether the differences in microglia reactivity may explain the differential susceptibility of different brain areas to insults, and particularly the higher sensitivity of CA1 pyramidal neurons to inflammatory stimuli. Understanding the spatiotemporal heterogeneity of microglia phenomics in health and disease is of paramount importance to find new druggable targets for the development of novel microglia-targeted therapies in different CNS disorders. This will allow interventions in three different ways: (i) by suppressing the pro-inflammatory properties of microglia to limit the deleterious effect of their activation; (ii) by modulating microglia phenotypic change to favor anti-inflammatory properties; (iii) by influencing microglia priming early in the disease process.

Laser emission at 675 nm: In vitro study evidence of a promising role in skin rejuvenation.

Light-based therapies have been proven to influence and perhaps reverse skin ageing at clinical, molecular and histologic levels. Laser technology decreases photodamage by promoting collagen type I and III synthesis and enhancing the expression of heat shock protein. Aims: This study aims to assess different doses of 675 nm irradiation on human dermal fibroblast cells to evaluate the potential therapeutic effects on the rejuvenation process. Methods: This study employed a laser system that emits 675 nm wavelength: 260, 390, 520 and 650 J/cm2 doses were tested on adult human dermal fibroblast cells. Cellular viability, proliferation, and synthesis of type I and III collagen were studied. Results: No dose tested showed effects on cell viability and proliferation at 24 and 48 h from the irradiation. Doses of 260 and 520 J/cm2 causes a significant decrease in type I collagen fluorescence intensity, while 390 J/cm2 elicits a significant increase in type III collagen expression. Conclusions: Our results showed that 675 nm laser irradiation does not affect cell viability while modulating cell proliferation and collagen synthesis in human adult cultured fibroblasts in vitro. These findings suggest that 675 nm laser irradiation potentially plays a role in skin rejuvenation.

Cannabidiol inhibits microglia activation and mitigates neuronal damage induced by kainate in an in-vitro seizure model.

BACKGROUND: Epilepsy is one of the most common brain disorder and, despite the possible use of several therapeutic options, many patients continue to have seizures for their entire lifespan and they need new therapeutic approaches. In the last years the interest on the non-psychoactive compounds present in Cannabis sativa has massively increased, and cannabidiol (CBD) has been shown to be effective in the treatment of different types of neurological disorders and neurodegenerative diseases such as epilepsy, ischemia, multiple sclerosis and Alzheimer's Disease. METHODS: We investigated the effects of the selected cannabinoids, Δ9-tetrahydrocannabinol (THC), CBD and cannabigerol (CBG) in rat organotypic hippocampal slices exposed to kainate, an in vitro seizure model. Cell death in the cornu Ammonis 3 (CA3) hippocampal subregion was quantified by propidium iodide fluorescence. Morphological analysis and tissue organization were examined by immunohistochemistry and confocal microscopy and microglia activation and polarization was evaluated using flow cytometry and morphology analysis. RESULTS: When present in the incubation medium, cannabidiol reduced dose-dependent CA3 injury induced by kainate. Conversely, incubation with THC exacerbated hippocampal damage. The neuroprotective effects of cannabidiol were blocked by TRPV1, TRPV2, 5-HT1A, and PPARγ antagonists. Confocal microscopy confirmed that CBD but not THC had a significant protective effect against neuronal damage and tissue disorganization caused by kainate. Cannabidiol incubation significantly block the microglia activation from the M0 to M1 phenotype observed in the kainate in-vitro seizure model, pushing toward a transition from M0 to M2. CONCLUSIONS: Our results suggest that CBD mitigated neuronal damage induced by kainate and blocked the transition from the M0 to the M1 phenotype.

The Protective Effect of CBD in a Model of In Vitro Ischemia May Be Mediated by Agonism on TRPV2 Channel and Microglia Activation.

Cannabinoids, used for centuries for recreational and medical purposes, have potential therapeutic value in stroke treatment. Cannabidiol (CBD), a non-psychoactive compound and partial agonist of TRPV2 channels, is efficacious in many neurological disorders. We investigated the effects of CBD or Δ9-tetrahydrocannabinol (THC) in rat organotypic hippocampal slices exposed to oxygen-glucose deprivation (OGD), an in vitro model of ischemia. Neuronal TRPV2 expression decreased after OGD, but it increased in activated, phagocytic microglia. CBD increased TRPV2 expression, decreased microglia phagocytosis, and increased rod microglia after OGD. THC had effects contrary to those of CBD. Our results show that cannabinoids have different effects in ischemia. CBD showed neuroprotective effects, mediated, at least in part, by TRPV2 channels, since the TRPV2 antagonist tranilast blocked them, while THC worsened the neurodegeneration caused by ischemia. In conclusion, our results suggest that different cannabinoid molecules play different roles in the mechanisms of post-ischemic neuronal death. These different effects of cannabinoid observed in our experiments caution against the indiscriminate use of cannabis or cannabinoid preparations for recreational or therapeutic use. It was observed that the positive effects of CBD may be counteracted by the negative effects caused by high levels of THC.

Blue-LED-Light Photobiomodulation of Inflammatory Responses and New Tissue Formation in Mouse-Skin Wounds.

Background: Recent studies evidence that blue-LED-light irradiation can modulate cell responses in the wound healing process within 24 h from treatment. This study aims to investigate blue-light (410-430 nm) photobiomodulation used in a murine wound model within six days post-treatment. Methods: A superficial wound was made in 30 CD1 male mice. The injuries were treated with a blue LED light (20.6 J/cm2), and biopsies were collected at 24, 72, and 144 h. Histology, fluorescence analysis, and advanced microscopy techniques were used. Results: We can observe an increase in the cellular infiltrate response, and in mast-cell density and their degranulation index correlated to the expression of the major histocompatibility complex after 24 h. Furthermore, after six days, the vessel density increases with the expression of the platelet-derived growth factor in the mast cells. Finally, collagen deposition and morphology in the treated wounds appear more similar to unwounded skin. Conclusions: Blue-light photobiomodulation stimulates several cellular processes that are finely coordinated by mast cells, leading to more rapid wound healing and a better-recovered skin morphology.

Synergistic Sequential Emission of Fractional 10.600 and 1540 nm Lasers for Skin Resurfacing: An Ex Vivo Histological Evaluation.

Background: Fractional ablative and non-ablative lasers are useful treatments for skin rejuvenation. A procedure that provides the sequential application of fractional ablative followed by non-ablative laser treatment may reduce patients' downtime and deliver better cosmetic results than with either laser alone. Objective: The purpose of the current study was to demonstrate the ameliorative and therapeutic effects in skin remodeling of the synergistic use of the two laser wavelengths (fractional ablative CO2 and non-ablative 1540 nm) with three different types of pulse shapes, S-Pulse (SP), D-Pulse (DP) and H-Pulse (HP), through which the CO2 laser can emit, performing an ex vivo histological evaluation. Methods: In this prospective study, ex vivo sheep inner thigh skin was chosen due to its similarity to human skin tissue, and a histological evaluation was performed. Three irradiation conditions, using all of the three CO2 pulse shapes (alone or averaged), were investigated: (1) 10.600 nm alone, the sequential irradiation of the two wavelengths in the same perfectly controlled energy pulses (DOT) for the entire scan area; ((2) 10.600 nm followed immediately by 1540 nm; and (3) 1540 nm followed immediately by 10.600 nm). Results: When comparing ablative to sequential irradiations, the synergy of the two wavelengths did not alter the typical ablative pulse shape of the 10.600 nm laser alone. With the same CO2 pulse shape, the lesion depth did not vary with the synergy of the two wavelengths, while thermal lesion width increased compared to CO2 alone. The ablation rate was achieved, while the total thermal lesion coverage in the scanning area of CO2 - 1540 lasers was greater than when using CO2 alone and then the other sequential irradiation. Conclusions: This study provides important preclinical data for new and early uses of the novel 10.600/1540 nm dual-wavelength non-ablative fractional laser. The synergy of the two wavelengths enhanced all the benefits already available when using CO2 laser systems both in terms of tone strengthening, thanks to a greater shrinking effect, and in terms of stimulation and collagen remodeling thanks to a greater volumetric thermal effect.

Cyanine-Doped Nanofiber Mats for Laser Tissue Bonding.

In spite of an extensive body of academic initiatives and innovative products, the toolkit of wound dressing has always revolved around a few common concepts such as adhesive patches and stitches and their variants. Our work aims at an alternative solution for an immediate restitutio ad integrum of the mechanical functionality in cutaneous repairs. We describe the fabrication and the application of electrospun mats of bioactive nanofibers all made of biocompatible components such as a natural polysaccharide and a cyanine dye for use as laser-activatable plasters, resembling the ultrastructure of human dermis. In particular, we investigate their morphological features and mechanical moduli under conditions of physiological relevance, and we test their use to bind a frequent benchmark of connective tissue as rabbit tendon and a significant case of clinical relevance as human dermis. Altogether, our results point to the feasibility of a new material for wound dressing combining translational potential, strength close to human dermis, extensibility exceeding 15% and state-of-art adhesive properties.

Hypoxia/Ischemia-Induced Rod Microglia Phenotype in CA1 Hippocampal Slices.

The complexity of microglia phenotypes and their related functions compels the continuous study of microglia in diseases animal models. We demonstrated that oxygen-glucose deprivation (OGD) induced rapid, time- and space-dependent phenotypic microglia modifications in CA1 stratum pyramidalis (SP) and stratum radiatum (SR) of rat organotypic hippocampal slices as well as the degeneration of pyramidal neurons, especially in the outer layer of SP. Twenty-four h following OGD, many rod microglia formed trains of elongated cells spanning from the SR throughout the CA1, reaching the SP outer layer where they acquired a round-shaped amoeboid phagocytic head and phagocytosed most of the pyknotic, damaged neurons. NIR-laser treatment, known to preserve neuronal viability after OGD, prevented rod microglia formation. In CA3 SP, pyramidal neurons were less damaged, no rod microglia were found. Thirty-six h after OGD, neuronal damage was more pronounced in SP outer and inner layers of CA1, rod microglia cells were no longer detectable, and most microglia were amoeboid/phagocytic. Damaged neurons, more numerous 36 h after OGD, were phagocytosed by amoeboid microglia in both inner and outer layers of CA1. In response to OGD, microglia can acquire different morphofunctional phenotypes which depend on the time after the insult and on the subregion where microglia are located.

Characterization and Ex Vivo Application of Indocyanine Green Chitosan Patches in Dura Mater Laser Bonding.

Dura mater repair represents a final and crucial step in neurosurgery: an inadequate dural reconstruction determines dreadful consequences that significantly increase morbidity and mortality rates. Different dural substitutes have been used with suboptimal results. To overcome this issue, in previous studies, we proposed a laser-based approach to the bonding of porcine dura mater, evidencing the feasibility of the laser-assisted procedure. In this work, we present the optimization of this approach in ex vivo experiments performed on porcine dura mater. An 810-nm continuous-wave AlGaAs (Aluminium Gallium Arsenide) diode laser was used for welding Indocyanine Green-loaded patches (ICG patches) to the dura. The ICG-loaded patches were fabricated using chitosan, a resistant, pliable and stable in the physiological environment biopolymer; moreover, their absorption peak was very close to the laser emission wavelength. Histology, thermal imaging and leak pressure tests were used to evaluate the bonding effect. We demonstrated that the application of 3 watts (W), pulsed mode (Ton 30 ms, Toff 3.5 ms) laser light induces optimal welding of the ICG patch to the dura mater, ensuring an average fluid leakage pressure of 216 ± 105 mmHg, falling within the range of physiological parameters. This study demonstrated that the thermal effect is limited and spatially confined and that the laser bonding procedure can be used to close the dura mater. Our results showed the effectiveness of this approach and encourage further experiments in in vivo models.

Photobiomodulation of Human Fibroblasts and Keratinocytes with Blue Light: Implications in Wound Healing.

In recent years, photobiomodulation (PBM) has been recognized as a physical therapy in wound management. Despite several published research papers, the mechanism underlying photobiomodulation is still not completely understood. The investigation about application of blue light to improve wound healing is a relatively new research area. Tests in selected patients evidenced a stimulation of the healing process in superficial and chronic wounds treated with a blue LED light emitting at 420 nm; a study in animal model pointed out a faster healing process in superficial wound, with an important role of fibroblasts and myofibroblasts. Here, we present a study aiming at evidencing the effects of blue light on the proliferation and metabolism in fibroblasts from healthy skin and keratinocytes. Different light doses (3.43, 6.87, 13.7, 20.6, 30.9 and 41.2 J/cm2) were used to treat the cells, evidencing inhibitory and stimulatory effects following a biphasic dose behavior. Electrophysiology was used to investigate the effects on membrane currents: healthy fibroblasts and keratinocytes showed no significant differences between treated and not treated cells. Raman spectroscopy revealed the mitochondrial Cytochrome C (Cyt C) oxidase dependence on blue light irradiation: a significant decrease in peak intensity of healthy fibroblast was evidenced, while it is less pronounced in keratinocytes. In conclusion, we observed that the blue LED light can be used to modulate metabolism and proliferation of human fibroblasts, and the effects in wound healing are particularly evident when studying the fibroblasts and keratinocytes co-cultures.

Experimental Study on Blue Light Interaction with Human Keloid-Derived Fibroblasts.

Keloids are an exuberant response to wound healing, characterized by an exaggerated synthesis of collagen, probably due to the increase of fibroblasts activity and to the reduction of their apoptosis rate: currently no standard treatments or pharmacological therapies are able to prevent keloid recurrence. To reach this goal, in recent years some physical treatments have been proposed, and among them the PhotoBioModulation therapy (PBM). This work analyses the effects of a blue LED light irradiation (410-430 nm, 0.69 W/cm2 power density) on human fibroblasts, isolated from both keloids and perilesional tissues. Different light doses (3.43-6.87-13.7-20.6-30.9 and 41.2 J/cm2) were tested. Biochemical assays and specific staining were used to assess cell metabolism, proliferation and viability. Micro-Raman spectroscopy was used to explore direct effects of the blue LED light on the Cytochrome C (Cyt C) oxidase. We also investigated the effects of the irradiation on ionic membrane currents by patch-clamp recordings. Our results showed that the blue LED light can modulate cell metabolism and proliferation, with a dose-dependent behavior and that these effects persist at least till 48 h after treatment. Furthermore, we demonstrated that the highest fluence value can reduce cell viability 24 h after irradiation in keloid-derived fibroblasts, while the same effect is observed 48 h after treatment in perilesional fibroblasts. Electrophysiological recordings showed that the medium dose (20.6 J/cm2) of blue LED light induces an enhancement of voltage-dependent outward currents elicited by a depolarizing ramp protocol. Overall, these data demonstrate the potentials that PBM shows as an innovative and minimally-invasive approach in the management of hypertrophic scars and keloids, in association with current treatments.

Optically activated and interrogated plasmonic hydrogels for applications in wound healing.

We disclose the use of hybrid materials featuring Au/Ag core/shell nanorods in porous chitosan/polyvinyl alcohol scaffolds for applications in tissue engineering and wound healing. The combination of Au and Ag in a single construct provides synergistic opportunities for optical activation of functions as near infrared laser tissue bonding, and remote interrogation to return parameters of prognostic relevance in wound healing monitoring. In particular, the bimetallic component ensures optical tunability, enhanced shelf life and photothermal stability, serves as a reservoir of germicidal silver cations, and changes in near-infrared and visible color according to the environmental level of oxidative stress. At the same time, the polymeric blend is ideal to bind connective tissue upon photothermal activation, and to support fabrication processes that provide high porosity, such as electrospinning, thus putting all the premises for cellular repopulation and antimicrobial protection.

Adenosine A2B receptors inhibit K+ currents and cell differentiation in cultured oligodendrocyte precursor cells and modulate sphingosine-1-phosphate signaling pathway.

Oligodendrocytes are the only myelinating cells in the brain and differentiate from their progenitors (OPCs) throughout adult life. However, this process fails in demyelinating pathologies. Adenosine is emerging as an important player in OPC differentiation and we recently demonstrated that adenosine A2A receptors inhibit cell maturation by reducing voltage-dependent K+ currents. No data are available to date about the A2B receptor (A2BR) subtype. The bioactive lipid mediator sphingosine-1-phosphate (S1P) and its receptors (S1P1-5) are also crucial modulators of OPC development. An interaction between this pathway and the A2BR is reported in peripheral cells. We studied the role of A2BRs in modulating K+ currents and cell differentiation in OPC cultures and we investigated a possible interplay with S1P signaling. Our data indicate that the A2BR agonist BAY60-6583 and its new analogue P453 inhibit K+ currents in cultured OPC and the effect was prevented by the A2BR antagonist MRS1706, by K+ channel blockers and was differently modulated by the S1P analogue FTY720-P. An acute (10 min) exposure of OPCs to BAY60-6583 also increased the phosphorylated form of sphingosine kinase 1 (SphK1). A chronic (7 days) treatment with the same agonist decreased OPC differentiation whereas SphK1/2 inhibition exerted the opposite effect. Furthermore, A2BR was overexpressed during OPC differentiation, an effect prevented by the pan SphK1/2 inhibitor VPC69047. Finally, A2BR silenced cells showed increased cell maturation, decreased SphK1 expression and enhanced S1P lyase levels. We conclude that A2BRs inhibit K+ currents and cell differentiation and positively modulate S1P synthesis in cultured OPCs.

Protective Effect of Adenosine A2B Receptor Agonist, BAY60-6583, Against Transient Focal Brain Ischemia in Rat.

Cerebral ischemia is a multifactorial pathology characterized first by an acute injury, due to excitotoxicity, followed by a secondary brain injury that develops hours to days after ischemia. During ischemia, adenosine acts as an endogenous neuroprotectant. Few studies have investigated the role of A2B receptor in brain ischemia because of the low potency of adenosine for it and the few selective ligands developed so far. A2B receptors are scarcely but widely distributed in the brain on neurons, glial and endothelial cells and on hematopoietic cells, lymphocytes and neutrophils, where they exert mainly anti-inflammatory effects, inhibiting vascular adhesion and inflammatory cells migration. Aim of this work was to verify whether chronic administration of the A2B agonist, BAY60-6583 (0.1 mg/kg i.p., twice/day), starting 4 h after focal ischemia induced by transient (1 h) Middle Cerebral Artery occlusion (tMCAo) in the rat, was protective after the ischemic insult. BAY60-6583 improved the neurological deficit up to 7 days after tMCAo. Seven days after ischemia BAY60-6583 reduced significantly the ischemic brain damage in cortex and striatum, counteracted ischemia-induced neuronal death, reduced microglia activation and astrocytes alteration. Moreover, it decreased the expression of TNF-α and increased that of IL-10 in peripheral plasma. Two days after ischemia BAY60-6583 reduced blood cell infiltration in the ischemic cortex. The present study indicates that A2B receptors stimulation can attenuate the neuroinflammation that develops after ischemia, suggesting that A2B receptors may represent a new interesting pharmacological target to protect from degeneration after brain ischemia.

Hybrid organosilicon/polyol phantom for photoacoustic imaging.

The rapid development of hardware and software for photoacoustic technologies is urging the establishment of dedicated tools for standardization and performance assessment. In particular, the fabrication of anatomical phantoms for photoacoustic imaging remains an open question, as current solutions have not yet gained unanimous support. Here, we propose that a hybrid material made of a water-in-oil emulsion of glycerol and polydimethylsiloxane may represent a versatile platform to host a broad taxonomy of hydrophobic and hydrophilic dyes and recapitulate the optical and acoustic features of bio tissue. For a full optical parameterization, we refer to Wróbel, et al. [ Biomed. Opt. Express7, 2088 (2016)], where this material was first presented for optical imaging. Instead, here, we complete the picture and find that its speed of sound and acoustic attenuation resemble those of pure polydimethylsiloxane, i.e. respectively 1150 ± 30 m/s and 3.5 ± 0.4 dB/(MHz·cm). We demonstrate its use under a commercial B-mode scanner and a home-made A-mode stage for photoacoustic analysis to retrieve the ground-truth encoded in a multilayer architecture containing indocyanine green, plasmonic particles and red blood cells. Finally, we verify the stability of its acoustic, optical and geometric features over a time span of three months.