Assessment of skin sensitization properties of few-layer graphene and graphene oxide through the Local Lymph Node Assay (OECD TG 442B).

Skin contact is one of the most common exposure routes to graphene-based materials (GBMs) during their small-scale and industrial production or their use in technological applications. Nevertheless, toxic effects in humans by cutaneous exposure to GBMs remain largely unexplored, despite skin contact to other related materials has been associated with adverse effects. Hence, this in vivo study was carried out to evaluate the cutaneous effects of two GBMs, focusing on skin sensitization as a possible adverse outcome. Skin sensitization by few-layer graphene (FLG) and graphene oxide (GO) was evaluated following the Organization for Economic Cooperation and Development (OECD) guideline 442B (Local Lymph Node Assay; LLNA) measuring the proliferation of auricular lymph node cells during the induction phase of skin sensitization. Groups of four female CBA/JN mice (8-12 weeks) were daily exposed to FLG or GO through the dorsal skin of each ear (0.4-40 mg/mL, equal to 0.01-1.00 mg/ear) for 3 consecutive days, and proliferation of auricular lymph node cells was evaluated 3 days after the last treatment. During this period, no clinical signs of toxicity and no alterations in body weight and food or water consumptions were observed. In addition, no ear erythema or edema were recorded as signs of irritation or inflammation. Bromo-deoxyuridine (BrdU) incorporation in proliferating lymphocytes from ear lymph nodes (stimulation indexes <1.6) and the histological analysis of ear tissues excluded sensitizing or irritant properties of these materials, while myeloperoxidase activity in ear biopsies confirmed no inflammatory cells infiltrate. On the whole, this study indicates the absence of sensitization and irritant potential of FLG and GO.

Acute Toxicity by Oral Co-Exposure to Palytoxin and Okadaic Acid in Mice.

The frequent occurrence of marine dinoflagellates producing palytoxin (PLTX) or okadaic acid (OA) raises concern for the possible co-presence of these toxins in seafood, leading to additive or synergistic adverse effects in consumers. Thus, the acute oral toxicity of PLTX and OA association was evaluated in mice: groups of eight female CD-1 mice were administered by gavage with combined doses of PLTX (30, 90 or 270 µg/kg) and OA (370 µg/kg), or with each individual toxin, recording signs up to 24 h (five mice) and 14 days (three mice). Lethal effects occurred only after PLTX (90 or 270 µg/kg) exposure, alone or combined with OA, also during the 14-day recovery. PLTX induced scratching, piloerection, abdominal swelling, muscle spasms, paralysis and dyspnea, which increased in frequency or duration when co-administered with OA. The latter induced only diarrhea. At 24 h, PLTX (90 or 270 µg/kg) and OA caused wall redness in the small intestine or pale fluid accumulation in its lumen, respectively. These effects co-occurred in mice co-exposed to PLTX (90 or 270 µg/kg) and OA, and were associated with slight ulcers and inflammation at forestomach. PLTX (270 µg/kg alone or 90 µg/kg associated with OA) also decreased the liver/body weight ratio, reducing hepatocyte glycogen (270 µg/kg, alone or combined with OA). No alterations were recorded in surviving mice after 14 days. Overall, the study suggests additive effects of PLTX and OA that should be considered for their risk assessment as seafood contaminants.

Ecotoxicological Impact of the Marine Toxin Palytoxin on the Micro-Crustacean Artemia franciscana.

Palytoxin (PLTX) is a highly toxic polyether identified in various marine organisms, such as Palythoa soft corals, Ostreopsis dinoflagellates, and Trichodesmium cyanobacteria. In addition to adverse effects in humans, negative impacts on different marine organisms have been often described during Ostreopsis blooms and the concomitant presence of PLTX and its analogues. Considering the increasing frequency of Ostreopsis blooms due to global warming, PLTX was investigated for its effects on Artemia franciscana, a crustacean commonly used as a model organism for ecotoxicological studies. At concentrations comparable to those detected in culture media of O. cf. ovata (1.0-10.0 nM), PLTX significantly reduced cysts hatching and induced significant mortality of the organisms, both at larval and adult stages. Adults appeared to be the most sensitive developmental stage to PLTX: significant mortality was recorded after only 12 h of exposure to PLTX concentrations > 1.0 nM, with a 50% lethal concentration (LC50) of 2.3 nM (95% confidence interval = 1.2-4.7 nM). The toxic effects of PLTX toward A. franciscana adults seem to involve oxidative stress induction. Indeed, the toxin significantly increased ROS levels and altered the activity of the major antioxidant enzymes, in particular catalase and peroxidase, and marginally glutathione-S-transferase and superoxide dismutase. On the whole, these results indicate that environmentally relevant concentrations of PLTX could have a negative effect on Artemia franciscana population, suggesting its potential ecotoxicological impact at the marine level.

Partial Reversibility of the Cytotoxic Effect Induced by Graphene-Based Materials in Skin Keratinocytes.

In the frame of graphene-based material (GBM) hazard characterization, particular attention should be given to the cutaneous effects. Hence, this study investigates if HaCaT skin keratinocytes exposed to high concentrations of few-layer graphene (FLG) or partially dehydrated graphene oxide (d-GO) for a short time can recover from the cytotoxic insult, measured by means of cell viability, mitochondrial damage and oxidative stress, after GBM removal from the cell medium. When compared to 24 or 72 h continuous exposure, recovery experiments suggest that the cytotoxicity induced by 24 h exposure to GBM is only partially recovered after 48 h culture in GBM-free medium. This partial recovery, higher for FLG as compared to GO, is not mediated by autophagy and could be the consequence of GBM internalization into cells. The ability of GBMs to be internalized inside keratinocytes together with the partial reversibility of the cellular damage is important in assessing the risk associated with skin exposure to GBM-containing devices.

Through-the-Wall Microwave Imaging: Forward and Inverse Scattering Modeling.

The imaging of dielectric targets hidden behind a wall is addressed in this paper. An analytical solver for a fast and accurate computation of the forward scattered field by the targets is proposed, which takes into account all the interactions of the electromagnetic field with the interfaces of the wall. Furthermore, an inversion procedure able to address the full underlying non-linear inverse scattering problem is introduced. This technique exploits a regularizing scheme in Lebesgue spaces in order to reconstruct an image of the hidden targets. Preliminary numerical results are provided in order to initially assess the capabilities of the developed solvers.

Skin irritation potential of graphene-based materials using a non-animal test.

Besides inhalation, skin contact may be considered one of the most relevant exposure routes to graphene-based materials (GBMs). However, very few data on the cutaneous toxicity of these materials are available, so far. This study is focused on skin irritation potential of a panel of GBMs: few-layer graphene (FLG), exfoliated by ball milling of graphite, FLG exfoliated by ultrasonication using sodium dodecyl sulfate (FLG-SDS) or sodium dodecylbenzenesulfonate (FLG-SDBS), CVD-graphene, obtained by chemical vapor deposition, graphene oxide (GO) and reduced GO (rGO). Skin irritation was assessed using the SkinEthic™ Reconstructed human Epidermis (RhE), following the Organisation for Economic Co-operation and Development (OECD) Test Guideline (TG) 439. Even though not validated for nanomaterials, the OCED TG 439 turned out to be applicable also for GBM testing, since no interference with the methylthiazolyldiphenyl-tetrazolium bromide (MTT) reduction, used as a final readout, was found. Furthermore, direct epidermal exposure to powdered GBMs mimics the actual human exposure, avoiding interference by the cell culture medium (protein corona formation). Only GBMs prepared with irritant surfactants (FLG-SDS and FLG-SDBS), but not the others, reduced RhE viability at levels lower than those predicting skin irritation (≤50%), suggesting irritant properties. This result was further confirmed by measuring cytokine (IL-1α, IL-6 and IL-8) release by GBM-treated RhE and by histological analysis as additional readouts to implement the guideline. On the whole, these results demonstrate that GBMs prepared with non-irritant exfoliation agents do not induce skin irritation after a single acute exposure.

Interferon gamma inducible protein 16 (IFI16) expression is reduced in mantle cell lymphoma.

IFI16, member of the IFN-inducible PYHIN-200 gene family, modulates proliferation, survival and differentiation of different cell lineages. In particular, IFI16 expression, which is regulated during the differentiation of B cells, was recently studied in B-CLL as well. Here, we compared IFI16 expression in several lymphomas including Burkitt lymphoma, diffuse large B-cell lymphoma, follicular lymphoma, marginal zone lymphoma and mantle cell lymphoma with respect to normal cell counterparts. We observed that IFI16 expression was significantly deregulated only in mantle cell lymphoma (p < 0.05). Notably, IFI16 was associated with the expression of genes involved in interferon response, cell cycle, cell death and proliferation and, interestingly, lipid and glucose metabolism, suggesting that IFI16 deregulation might be associated with relevant changes in cell biology. In our group of mantle cell lymphoma samples a correlation between patient survival and IFI16 expression was not detected even though mantle cell lymphoma prognosis is known to be associated with cell proliferation. Altogether, these results suggest a complex relationship between IFI16 expression and MCL which needs to be analyzed in further studies.

IFI16 reduced expression is correlated with unfavorable outcome in chronic lymphocytic leukemia.

Chronic lymphocytic leukemia (CLL) is the most common leukemia in adults. Its clinical course is typically indolent; however, based on a series of pathobiological, clinical, genetic, and phenotypic parameters, patient survival varies from less than 5 to more than 20 years. In this paper, we show for the first time that the expression of the interferon-inducible DNA sensor IFI16, a member of the PYHIN protein family involved in proliferation inhibition and apoptosis regulation, is associated with the clinical outcome in CLL. We studied 99 CLLs cases by immunohistochemistry and 10 CLLs cases by gene expression profiling. We found quite variable degrees of IFI16 expression among CLLs cases. Noteworthy, we observed that a reduced IFI16 expression was associated with a very poor survival, but only in cases with ZAP70/CD38 expression. Furthermore, we found that IFI16 expression was associated with a specific gene expression signature. As IFI16 can be easily detected by immunohistochemistry or flow cytometry, it may become a part of phenotypic screening in CLL patients if its prognostic role is confirmed in independent series.

On beam shaping of the field radiated by a line source coupled to finite or infinite photonic crystals.

Comparison of the beam-shaping effect of a field radiated by a line source, when an ideal infinite structure constituted by two photonic crystals and an actual finite one are considered, has been carried out by means of two different methods. The lattice sums technique combined with the generalized reflection matrix method is used to rigorously investigate the radiation from the infinite photonic crystals, whereas radiation from crystals composed of a finite number of rods along the layers is analyzed using the cylindrical-wave approach. A directive radiation is observed with the line source embedded in the structure. With an increased separation distance between the crystals, a significant edge diffraction appears that provides the main radiation mechanism in the finite layout. Suitable absorbers are implemented to reduce the above-mentioned diffraction and the reflections at the boundaries, thus obtaining good agreement between radiation patterns of a localized line source coupled to finite and infinite photonic crystals, when the number of periods of the finite structure is properly chosen.

Entrectinib, a Pan-TRK, ROS1, and ALK Inhibitor with Activity in Multiple Molecularly Defined Cancer Indications.

Activated ALK and ROS1 tyrosine kinases, resulting from chromosomal rearrangements, occur in a subset of non-small cell lung cancers (NSCLC) as well as other tumor types and their oncogenic relevance as actionable targets has been demonstrated by the efficacy of selective kinase inhibitors such as crizotinib, ceritinib, and alectinib. More recently, low-frequency rearrangements of TRK kinases have been described in NSCLC, colorectal carcinoma, glioblastoma, and Spitzoid melanoma. Entrectinib, whose discovery and preclinical characterization are reported herein, is a novel, potent inhibitor of ALK, ROS1, and, importantly, of TRK family kinases, which shows promise for therapy of tumors bearing oncogenic forms of these proteins. Proliferation profiling against over 200 human tumor cell lines revealed that entrectinib is exquisitely potent in vitro against lines that are dependent on the drug's pharmacologic targets. Oral administration of entrectinib to tumor-bearing mice induced regression in relevant human xenograft tumors, including the TRKA-dependent colorectal carcinoma KM12, ROS1-driven tumors, and several ALK-dependent models of different tissue origins, including a model of brain-localized lung cancer metastasis. Entrectinib is currently showing great promise in phase I/II clinical trials, including the first documented objective responses to a TRK inhibitor in colorectal carcinoma and in NSCLC. The drug is, thus, potentially suited to the therapy of several molecularly defined cancer settings, especially that of TRK-dependent tumors, for which no approved drugs are currently available. Mol Cancer Ther; 15(4); 628-39. ©2016 AACR.

Pro-inflammatory effects of palytoxin: an in vitro study on human keratinocytes and inflammatory cells.

Palytoxin (PLTX) is one of the most harmful marine toxins known so far. Although the ingestion of contaminated seafood is the most dangerous exposure route for humans, cutaneous and inhalational exposures are far more frequent, and can cause strong inflammatory reactions. However, little is known about the inflammatory events that follow the cutaneous exposure to the toxin. In this study, we investigated (1) the effects of both short (2 h) and long (24 h) term exposures of HaCaT keratinocytes to a sub-cytotoxic PLTX concentration on pro-inflammatory mediator gene expression and release and (2) the effect of PLTX-conditioned HaCaT cell media on undifferentiated (monocytes) and differentiated (macrophages; immature dendritic cells, iDCs; mature dendritic cells, mDCs) THP-1 cells. At 10-11 M, PLTX induced interleukin (IL)-6 and IL-8 release from HaCaT keratinocytes after 24 h of continuous exposure to the toxin, as well as after 23 h in toxin-free medium preceded by 1 h exposure to PLTX. Under the same experimental conditions, release of the inflammatory mediators prostaglandin-E2 and histamine was also found after both short and long exposures to the toxin. The conditioned media collected from HaCaT cells treated with PLTX increased the migration of the differentiated and undifferentiated THP-1 cells (potency rank order: monocytes ≥ iDCs > mDCs > macrophages) but did not induce cell differentiation. These results indicate that keratinocytes can be actively involved in the recruitment of inflammatory cells in response to cutaneous contact with PLTX. The lack of a significant effect on monocyte differentiation towards mature immune cells suggests that PLTX is endowed with irritant rather than sensitizing properties.

Calcaneal quantitative ultrasound (QUS) and dual X-ray absorptiometry (DXA) bone analysis in adult HIV-positive patients.

Human immunodeficiency virus (HIV)-infected patients have an increased risk of developing osteopenia or osteoporosis compared with healthy individuals. Our aim was to compare dual X-ray absorptiometry (DXA), the gold standard for measuring bone mineral density (BMD), with bone quantitative ultrasound (QUS), an alternative technique for predicting fractures and screening low BMD, at least in postmenopausal populations. We analyzed DXA and QUS parameters to investigate their accuracy in the diagnosis and prediction of bone alterations in a cohort of 224 HIV-1-positive patients. The speed of sound (SOS), broadband ultrasound attenuation (BUA) and stiffness index (SI) parameters showed a moderate correlation with DXA, especially with total-body BMD (r coefficient of 0.38, 0.4 and 0.42 respectively), particularly in the female subgroup. In addition, multivariate analysis of HIV-positive patients assessed for vertebral fractures indicated that QUS was more effective than DXA at predicting the risk of fracture. QUS can be used as an additional tool for analyzing bone density in HIV-positive patients and its case of use and low cost make it especially suitable for resource-limited settings where DXA is not employed.

The TPM3-NTRK1 rearrangement is a recurring event in colorectal carcinoma and is associated with tumor sensitivity to TRKA kinase inhibition.

The NTRK1 gene encodes Tropomyosin-related kinase A (TRKA), the high-affinity Nerve Growth Factor Receptor. NTRK1 was originally isolated from a colorectal carcinoma (CRC) sample as component of a somatic rearrangement (TPM3-NTRK1) resulting in expression of the oncogenic chimeric protein TPM3-TRKA, but there has been no subsequent report regarding the relevance of this oncogene in CRC. The KM12 human CRC cell line expresses the chimeric TPM3-TRKA protein and is hypersensitive to TRKA kinase inhibition. We report the detailed characterization of the TPM3-NTRK1 genomic rearrangement in KM12 cells and through a cellular screening approach, the identification of NMS-P626, a novel highly potent and selective TRKA inhibitor. NMS-P626 suppressed TPM3-TRKA phosphorylation and downstream signaling in KM12 cells and showed remarkable antitumor activity in mice bearing KM12 tumors. Finally, using quantitative reverse transcriptase PCR and immunohistochemistry (IHC) we identified the TPM3-NTRK1 rearrangement in a CRC clinical sample, therefore suggesting that this chromosomal translocation is indeed a low frequency recurring event in CRC and that such patients might benefit from therapy with TRKA kinase inhibitors.

Electromagnetic scattering by a circular cylinder buried below a slightly rough Gaussian surface.

A two-dimensional beam is scattered by a cylinder buried below a slightly rough surface. The cylindrical wave approach is applied, i.e., cylindrical waves are employed as basis functions of the fields scattered by the cylinder. Moreover, a spectral representation of both the incident field and the cylindrical waves is used. Rough surface deviation is coped with by the first-order small perturbation method. Therefore, to a zeroth-order solution relevant to scattering in the case of a flat surface, a first-order approximation is superimposed. The theoretical approach has been implemented for a periodic surface with Gaussian roughness spectrum.

Through-wall electromagnetic scattering by N conducting cylinders.

A spectral-domain analysis is presented for the scattering by perfectly conducting cylindrical objects behind a dielectric wall. The solution is developed with an analytical-numerical technique, based on the cylindrical wave approach. Suitable cylindrical functions and their spectral representations are introduced as basis functions for the scattered fields, to deal with their interaction with the planar interfaces bounding the wall. The numerical solution is given in TE and TM polarizations states, and in both near- and far-field zones. The model yields an accurate computation of direct scattering that can be useful for through-wall-imaging applications. A stack of three different dielectric media is considered in the theoretical model. In the numerical results, the upper medium, where the incident field is generated, is assumed to be filled by air, the central layer represents the wall, and the lower medium, which contains the scatterers, is air filled, too. Also general problems of scattering by buried objects can be simulated, being the cylinders buried in a medium of arbitrary permittivity, placed below a dielectric layer.

Effects of human immunodeficiency virus on the erythrocyte and megakaryocyte lineages.

Anaemia and thrombocytopenia are haematological disorders that can be detected in many human immunodeficiency virus (HIV)-positive patients during the development of HIV infection. The progressive decline of erythrocytes and platelets plays an important role both in HIV disease progression and in the clinical and therapeutic management of HIV-positive patients. HIV-dependent impairment of the megakaryocyte and erythrocyte lineages is multifactorial and particularly affects survival, proliferation and differentiation of bone marrow (BM) CD34+ haematopoietic progenitor cells, the activity of BM stromal cells and the regulation of cytokine networks. In this review, we analyse the major HIV-related mechanisms that are involved in the genesis and development of the anaemia and thrombocytopenia observed in HIV positive patients.

HIV-related mechanisms in atherosclerosis and cardiovascular diseases.

HIV-infected patients have a significantly higher risk of developing cardiovascular events during the progression of HIV disease. Atherosclerosis, myocardial infarction, cerebrovascular injury, pulmonary hypertension and thrombosis are consistently described in both combined antiretroviral therapy (cART)-treated and naive HIV-positive patients as major clinical complications. Recent studies indicate that the pathogenesis of cardiovascular lesions in HIV-positive patients is related to direct and indirect effects of HIV infection on vessel structures, independently of traditional risk factors. HIV infection strongly interferes with the biology of several cellular targets such as macrophage and endothelial cells. Moreover, HIV induces a profound derangement of lipid metabolism and inflammatory cytokine networks that are directly involved in atherogenesis and progressive impairment of the cardiovascular system.In this review, we discuss these major HIV-related mechanisms able to promote atherosclerosis and cardiovascular diseases in HIV-positive patients.

Oxidative stress induced by palytoxin in human keratinocytes is mediated by a H+-dependent mitochondrial pathway.

In the last decades, massive blooms of palytoxin (PLTX)-producing Ostreopsis cf. ovata have been observed along Mediterranean coasts, usually associated to human respiratory and cutaneous problems. At the molecular level, PLTX induces a massive intracellular Na(+) influx due to the transformation of Na(+)/K(+) ATPase in a cationic channel. Recently, we have demonstrated that Na(+) overload is the crucial step in mediating overproduction of reactive oxygen species (ROS) and cell death in human HaCaT keratinocytes, tentatively explaining PLTX-induced skin irritant effects. In the present study the molecular mechanisms of ROS production induced by PLTX-mediated Na(+) intracellular overload have been investigated. In HaCaT cells, PLTX exposure caused accumulation of superoxide anion, but not of nitric oxide or peroxynitrite/hydroxyl radicals. Even if RT-PCR and western blot analysis revealed an early NOX-2 and iNOS gene and protein over-expressions, their active involvement seemed to be only partial since selective inhibitors did not completely reduce O(2)(-) production. A significant role of other enzymes (COX-1, COX-2, XO) was not evidenced. Nigericin, that counteracts Na(+)-mediated H(+)-imbalance, dissipating ΔpH across mitochondrial inner membrane, and the uncouplers DNP significantly reduced O(2)(-) production. These inhibitions were synergistic when co-exposed with complex-I inhibitor rotenone. These results suggest a novel mechanism of O(2)(-) production induced by PLTX-mediated ionic imbalance. Indeed, the H(+) intracellular overload that follows PLTX-induced intracellular Na(+) accumulation, could enhance ΔpH across mitochondrial inner membrane, that seems to be the driving force for O(2)(-) production by reversing mitochondrial electron transport.

Analysis of the effects of HIV-1 Tat on the survival and differentiation of vessel wall-derived mesenchymal stem cells.

HIV infection is an independent risk factor for atherosclerosis development and cardiovascular damage. As vessel wall mesenchymal stem cells (MSCs) are involved in the regulation of vessel structure homeostasis, we investigated the role of Tat, a key factor in HIV replication and pathogenesis, in MSC survival and differentiation. The survival of subconfluent MSCs was impaired when Tat was added at high concentrations (200-1,000 ng/ml), whereas lower Tat concentrations (1-100 ng/ml) did not promote apoptosis. Tat enhanced the differentiation of MSC toward adipogenesis by the transcription and activity upregulation of PPARγ. This Tat-related modulation of adipogenesis was tackled by treatment with antagonists of Tat-specific receptors such as SU5416 and RGD Fc. In contrast, Tat inhibited the differentiation of MSCs to endothelial cells by downregulating the expression of VEGF-induced endothelial markers such as Flt-1, KDR, and vWF. The treatment of MSCs with Tat-derived peptides corresponding to the cysteine-rich, basic, and RGD domains indicated that these Tat regions are involved in the inhibition of endothelial marker expression. The Tat-related impairment of MSC survival and differentiation might play an important role in vessel damage and formation of the atherosclerotic lesions observed in HIV-infected patients.

Lignan derivatives from Krameria lappacea roots inhibit acute inflammation in vivo and pro-inflammatory mediators in vitro.

The roots of Krameria lappacea are used traditionally against oropharyngeal inflammation. So far, the astringent and antimicrobial properties of its proanthocyanidin constituents are considered to account for the anti-inflammatory effect. The aim of the present study was to characterize pharmacologically a lipophilic extract of K. lappacea roots and several isolated lignan derivatives (1-11) in terms of their putative anti-inflammatory activity. The dichloromethane extract (ID50 77 µg/cm²) as well compounds 1-11 (ID50 0.31-0.60 µmol/cm²) exhibited topical antiedematous properties comparable to those of indomethacin (ID50 0.29 µmol/cm²) in a mouse ear in vivo model. Two of the most potent compounds, 2-(2-hydroxy-4-methoxyphenyl)-5-(3-hydroxypropyl)benzofuran (5) and (+)-conocarpan (7), were studied regarding their time-dependent edema development and leukocyte infiltration up to 48 h after croton oil-induced dermatitis induction, and they showed activity profiles similar to that of hydrocortisone. In vitro studies of the isolated lignan derivatives demonstrated the inhibition of NF-κB, cyclooxygenase-1 and -2, 5-lipoxygenase, and microsomal prostaglandin E2 synthase-1 as well as antioxidant properties, as mechanisms possibly contributing to the observed in vivo effects. The present findings not only support the ethnopharmacological use of K. lappacea roots but also reveal that the isolated lignan derivatives contribute strongly to the anti-inflammatory activity of this herbal drug.