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Langerhans cell histiocytosis (LCH) is a rare disorder characterized by tissue accumulation of CD1a+CD207+ LCH cells. In LCH, somatic mutations of the BRAFV600E gene have been detected in tissue LCH cells, bone marrow CD34+ hematopoietic stem cells, circulating CD14+ monocytes, and BDCA1+ myeloid dendritic cells (DC). Targeting BRAFV600E in clonal Langerhans cells (LC) and their precursors is a potential treatment option for patients whose tumors have the mutation. The development of mouse macrophages and LCs is regulated by the CSF1 receptor (CSF1R). In patients with diffuse-type tenosynovial giant cell tumors, CSF1R inhibition depletes tumor-associated macrophages (TAM) with therapeutic efficacy; however, CSF1R signaling in LCs and LCH has not been investigated. We found through IHC and flow cytometry that CSF1R is normally expressed on human CD1a+CD207+ LCs in the epidermis and stratified epithelia. LCs that were differentiated from CD14+ monocytes, BDCA1+ DCs, and CD34+ cord blood progenitors expressed CSF1R that was downregulated upon maturation. Immature LCs migrated toward CSF1, but not IL34. Administration of the c-FMS/CSF1R kinase inhibitors GW2580 and BLZ945 significantly reduced human LC migration. In LCH clinical samples, LCH cells (including BRAFV600E cells) and TAMs retained high expression of CSF1R. We also detected the presence of transcripts for its ligand, CSF1, but not IL34, in all tested LCH cases. CSF1R and CSF1 expression in LCH, and their role in LC migration and differentiation, suggests CSF1R signaling blockade as a candidate rational approach for treatment of LCH, including the BRAFV600E and wild-type forms of the disease.

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Mesenchymal stem cells (MSCs) are multipotent cells, originally derived from the embryonic mesenchyme, and able to differentiate into connective tissues such as bone, fat, cartilage, tendon, and muscle. Furthermore, MSCs derived from adipose tissue ADSC (Adipose derived Stem Cells) show a great potential for degenerative diseases treatment. In this study, we designed a series of experiments based on real-time rt-QPCR to validated a commercially available kit able to explore changes in gene expression under osteogenic, adipogenic and chondrogenic differentiation of human ADSC. Initially, we selected a better indicators of trilineage differentiation by using third passages of cultured ADSC from stromal vascular fraction (SVF) isolated from fresh adipose tissue by enzymatic digestion. On the basis of statistically significant results ACAN, FABP4A and Col11a1 were chosen as indicators of chondrogenic, adipogenic and osteogenic differentiation respectively. An in-vitro aging analysis was then performed to evaluate the ADSC passage with the highest differentiation potential. Total RNA extraction from induced differentiation and controls ADSC from passage 2-6 and relative quantifications of mRNA expression of selected genes were performed according to rt-PCR kits tested. The chondrogenic differentiation test showed equivalent ∆∆Ct values for ACAN detection for cell passages ranging from P3 to P6, proving that they can be considered as equivalent samples for differentiation assays evaluation. For what concerns adipogenic differentiation and FABP4 detection, similar results were observed in all the cell passages tested; on the contrary only passage P6 showed suitable ∆∆Ct values for Col11a1 detection for osteogenic differentiation evaluation. In conclusion, we have validated a suitable real-time rt-QPCR protocol for osteogenic, chondrogenic and adipogenic ADSC differentiation ability evaluation in-vitro.

Multidrug-resistant (MDR) Gram-negative bacteria (GNB), such as Acinetobacter and Klebsiella, are responsible for severe hospital-acquired infections. Colistin, despite its toxicity and low tissue penetration, is considered the last resort antibiotic against these microorganisms. Of concern, the use of Colistin has recently been compromised by the emergence of Colistin resistance. Herein, we developed a new formulation consisting of multifunctional chitosan-coated human albumin nanoparticles for the delivery of Colistin (Col/haNPs). Col/haNPs were in vitro characterized for encapsulation efficiency, drug release, stability and cytotoxicity and were evaluated for antibacterial activity against MDR GNB (Acinetobacter baumannii and Klebsiella pneumoniae). Col/haNPs showed sizes lower than 200 nm, high encapsulation efficiency (98.65%) and prolonged in vitro release of Colistin. The safety of the nanoformulation was demonstrated by a negligible cytotoxicity on human fibroblasts and hemolytic activity. Col/haNPs evidenced a high antibacterial effect with a significant decrease in MIC values compared to free Colistin, in particular against Col-resistant strains with a pronounced decline of bacterial growth over time. Moreover, Col/haNPs exhibited an inhibitory effect on biofilm formation that was 4 and 60 fold higher compared to free Colistin, respectively for Colistin susceptible and resistant A. baumannii. Our findings suggest that Col/haNPs could represent a promising Colistin nanocarrier with high antimicrobial activity on MDR GNB.

Background: The dermis is a commonly used source tissue for biologic scaffolds; all cellular and nuclear materials need to be removed to limit the inflammatory immune response by the host organism. The decellularization is critical because it must preserve the structural integrity of the extracellular matrix. This work has analyzed a decellularization procedure commonly followed for the dermal tissue that is a chemical treatment with sodium hydroxide. The goal of this work is to identify the optimal treatment length on the basis of structural properties. Methods: Tensile tests have been performed on the native tissue and on tissues decellularized for 1-7 weeks in sodium hydroxide. The collected data have been analyzed through Tukey-Kramer test to assess if the mechanical properties (ultimate tensile stress and elastic modulus) of decellularized tissues were significantly different from the properties of the native tissue. These tests have been performed on specimens cut along two orthogonal directions (parallel and perpendicular to Langer’s lines). Results: The decellularization treatment performed with sodium hydroxide in general weakens the tissue: both the ultimate stress and the elastic modulus get lower. The structural properties along Langer lines orientation are more strongly impacted, while the structural properties orthogonal to Langer lines can be preserved with an optimal duration of the decellularization treatment that is 5-6 weeks. Conclusion: The duration of the decellularization treatment is critical not only to reach a complete decellularization, but also to preserve the mechanical properties of the tissue; 5-6 week treatment performed with sodium hydroxide allows preserving the mechanical properties of the native tissue perpendicularly to Langer lines orientation, and minimizing the impact of the decellularization process on the mechanical properties along the Langer lines orientation.

Osteoarthritis (OA) is characterized by articular cartilage degeneration and subchondral bone sclerosis. OA can benefit of non-surgical treatments with collagenase-isolated stromal vascular fraction (SVF) or cultured-expanded mesenchymal stem cells (ASCs). To avoid high manipulation of the lipoaspirate needed to obtain ASCs and SVF, we investigated whether articular infusions of autologous concentrated adipose tissue are an effective treatment for knee OA patients. The knee of 20 OA patients was intra-articularly injected with autologous concentrated adipose tissue, obtained after centrifugation of lipoaspirate. Patients’ articular functionality and pain were evaluated by VAS and WOMAC scores at three, six and 18 months from infusion. The osteogenic and chondrogenic ability of ASCs contained in the injected adipose tissue was studied in in vitro primary osteoblast and chondrocyte cell cultures, also plated on 3D-bone scaffold. Knee articular biopsies of patients previously treated with adipose tissue were analyzed. Immunohistochemistry (IHC) and scanning electron microscopy (SEM) were performed to detect cell differentiation and tissue regeneration. The treatment resulted safe, and all patients reported an improvement in terms of pain reduction and increase of function. According to the osteogenic or chondrogenic stimulation, ASCs expressed alkaline phosphatase or aggrecan, respectively. The presence of a layer of newly formed tissue was visualized by IHC staining and SEM. The biopsy of previously treated knee joints showed new tissue formation, starting from the bone side of the osteochondral lesion. Overall our data indicate that adipose tissue infusion stimulates tissue regeneration and might be considered a safe treatment for knee OA.

Mesenchymal stromal cells (MSCs) exert immunosuppressive effects on immune cells including dendritic cells (DCs). However, many details of the bidirectional interaction of MSCs with DCs are still unsolved and information on key molecules by which DCs can modulate MSC functions is limited. Here, we report that osteopontin (OPN), a cytokine involved in homeostatic and pathophysiologic responses, is constitutively expressed by DCs and regulated in the DC/MSC cocultures depending on the activation state of MSCs. Resting MSCs promoted OPN production, whereas the production of OPN was suppressed when MSCs were activated by proinflammatory cytokines (i.e., TNF-α, IL-6, and IL-1β). OPN induction required cell-to-cell contact, mediated at least in part, by β1 integrin (CD29). Conversely, activated MSCs inhibited the release of OPN via the production of soluble factors with a major role played by Prostaglandin E2 (PGE2). Accordingly, pretreatment with indomethacin significantly abrogated the MSC-mediated suppression of OPN while the direct addition of exogenous PGE2 inhibited OPN production by DCs. Furthermore, DC-conditioned medium promoted osteogenic differentiation of MSCs with a concomitant inhibition of adipogenesis. These effects were paralleled by the repression of the adipogenic markers PPARγ, adiponectin, and FABP4, and induction of the osteogenic markers alkaline phosphatase, RUNX2, and of the bone-anabolic chemokine CCL5. Notably, blocking OPN activity with RGD peptides or with an antibody against CD29, one of the OPN receptors, prevented the effects of DC-conditioned medium on MSC differentiation and CCL5 induction. Because MSCs have a key role in maintenance of bone marrow (BM) hematopoietic stem cell niche through reciprocal regulation with immune cells, we investigated the possible MSC/DC interaction in human BM by immunohistochemistry. Although DCs (CD1c+) are a small percentage of BM cells, we demonstrated colocalization of CD271+ MSCs with CD1c+ DCs in normal and myelodysplastic BM. OPN reactivity was observed in occasional CD1c+ cells in the proximity of CD271+ MSCs. Altogether, these results candidate OPN as a signal modulated by MSCs according to their activation status and involved in DC regulation of MSC differentiation.

Background The development of dermal scaffolds is of major interest in reconstructive surgery. Human Acellular Dermal Matrices (HADMs) provides biomechanical support and elicits new tissue formation. The use of allograft dermis is limited by its immunogenic characteristics. Our research group has focused on the use of human alloplastic glycerolized reticular dermis. Objective The dermal grafts were subjected to two different decellularization protocols in parallel, in order to compare the efficacy in the elimination of residual DNA. Methods It was compared the incubation of the dermis in NaOH (0.06 N) and in the standard culture medium "Dulbecco Modified Eagle Medium" (DMEM). The samples were incubated in the specific medium for 8 weeks. The newly developed real-time TaqMan® MGB-PCR assay was applied for both the detection and absolute quantification of residual DNA. Results It was observed that the level of residual DNA decreased until time T3 and remained constant until time T8. Moreover, there was no statistical difference between treatment with DMEM or NaOH 0.06 N as to the amount of residual DNA. Conclusions Decellularization methods, DMEM or NaOH 0.06 N do not affect DNA recovery. The proposed approach offers an alternative method to quantify residual DNA in HADM samples.

Growing warnings on health effects related to electronic cigarettes have met inconclusive findings at present. This study analyzed the in vitro percutaneous absorption of nicotine resulting by skin contamination with two e-liquids (refill 1 and 2) containing nicotine at 1.8%. Donor chambers of 6 Franz cells for each refill liquid were filled with 1 mL of nicotine e-liquid for 24 h; at selected intervals, 1.5 mL of the receptor solutions were collected for nicotine concentration analysis by mean gas chromatography–mass spectrometry (LOD: 0.01 μg/mL). The experiment was repeated removing the nicotine donor solution after 10 min from the application and rinsing the skin surface three times with 3.0 mL of milliQ water. A total of 12 cells with 24 h exposure and 12 cells washed were studied. The mean concentration of nicotine in the receiving phase at the end of the experiment was 54.9 ± 29.5 and 30.2 ± 18.4 μg/cm2 for refill 1 and 2 respectively and significantly lower in washed cells (4.7 ± 2.4 and 3.5 ± 1.3 μg/cm2). The skin absorption of nicotine can lead to minor health illness in vapers, while caution must be paid to dermal contamination by e liquids in children. The skin cleaning significantly reduced the transdermal absorption kinetic and intradermal deposition of nicotine.

Silver nanoparticles (AgNPs) are increasingly applied to a wide range of materials for biomedical use. These enable a close contact with human skin, thanks to the large release of silver ions that is responsible for a broad spectrum of antimicrobial activity. Silver can permeate the skin; however, there are no data available on silver permeation through skin grafts commonly used in burns recovery. The aim of our study was to evaluate silver penetration using fresh, cryopreserved, and glycerolized human skin grafts after exposure to a suspension of AgNPs in synthetic sweat using a Franz diffusion cell apparatus for 24 h. Silver permeation profiles revealed a significantly higher permeation through glycerolized skin compared with both fresh and cryopreserved skin: 24-h silver flux penetration was 0.2 ng cm(-2) h(-1) (lag time: 8.2 h) for fresh skin, 0.3 ng cm(-2) h(-1) (lag time: 10.9 h) for cryopreserved skin, and 3.8 ng cm(-2) h(-1) (lag time: 6.3 h) for glycerolized skin. Permeation through glycerolized skin is significantly higher compared to both fresh and cryopreserved skin. This result can generate relevant clinical implications for burns treatment with products containing AgNPs.

Osteoarthritis is characterized by loss of articular cartilage also due to reduced chondrogenic activity of mesenchymal stem cells (MSCs) from patients. Adipose tissue is an attractive source of MSCs (ATD-MSCs), representing an effective tool for reparative medicine, particularly for treatment of osteoarthritis, due to their chondrogenic and osteogenic differentiation capability. The treatment of symptomatic knee arthritis with ATD-MSCs proved effective with a single infusion, but multiple infusions could be also more efficacious. Here we studied some crucial aspects of adipose tissue banking procedures, evaluating ATD-MSCs viability, and differentiation capability after cryopreservation, to guarantee the quality of the tissue for multiple infusions. We reported that the presence of local anesthetic during lipoaspiration negatively affects cell viability of cryopreserved adipose tissue and cell growth of ATD-MSCs in culture. We observed that DMSO guarantees a faster growth of ATD-MSCs in culture than trehalose. At last, ATD-MSCs derived from fresh and cryopreserved samples at −80°C and −196°C showed viability and differentiation ability comparable to fresh samples. These data indicate that cryopreservation of adipose tissue at −80°C and −196°C is equivalent and preserves the content of ATD-MSCs in Stromal Vascular Fraction (SVF), guaranteeing the differentiation ability of ATD-MSCs.

The concentrated nicotine in e-cigarette refill liquids can be toxic if inadvertently ingested or absorbed through the skin. Reports of poisonings due to accidental ingestion of nicotine on refill liquids are rapidly increasing, while the evaluation of nicotine dermally absorbed still lacks. For that reason we studied transdermal nicotine absorption after the skin contamination with e-liquid. Donor chambers of eight Franz diffusion cells were filled with 1 mL of 0.8 mg/mL nicotine e-liquid for 24 h. The concentration of nicotine in the receiving phase was determined by high-performance liquid chromatography (LOD:0.1 μg/mL). Nicotine was detectable in receiving solution 2 h after the start of exposure and increased progressively. The medium flux calculated was 4.82 ± 1.05 μg/cm(2)/h with a lag time of 3.9 ± 0.1 h. After 24 h, the nicotine concentration in the receiving compartment was 101.02 ± 22.35 μg/cm(2) corresponding to 3.04 mg of absorbed nicotine after contamination of a skin surface of 100 cm(2). Skin contamination with e-liquid can cause nicotine skin absorption: caution must be paid when handling refill e-liquids.

Human acellular dermal matrices (HADMs) are used in reconstructive surgery as scaffolds promoting autologous tissue regeneration. Critical to the HADM ability to remodel and integrate into the host tissue is the removal of cells while maintaining an intact extracellular architecture. The objective of this work is to develop a methodology to analyse the mechanical properties of HADMs after decellularization to identify its ideal form of treatment and its duration. Two different decellularization techniques were used as a benchmark: the first is a well-established technique (incubation in NaOH for 1-7 weeks), and the second is an innovative technique developed by this research group (incubation in DMEM (Dulbecco's modified Eagle medium) for 1-7 weeks). After decellularization, the specimens underwent uniaxial tensile tests, and experimental data were represented with stress strain curves, calculating both engineering and true values. Mechanical tests have led to the identification of the optimal method (NaOH or DMEM) and duration for the decellularization treatment; differences between engineering and true values can reach 84%, but the engineering values remain useful to make comparisons, providing reliable indications with a simpler experimental set up and data processing.

Vitamin D receptor (VDR) mediates many genomic and non-genomic effects of vitamin D. Recently, the mitochondrial effects of vitamin D have been characterized in many cell types. In this article, we investigated the importance of VDR not only in mitochondrial activity and integrity but also in cell health. The silencing of the receptor in different healthy, non-transformed, and cancer cells initially decreased cell growth and modulated the cell cycle. We demonstrated that, in silenced cells, the increased respiratory activity was associated with elevated reactive oxygen species (ROS) production. In the long run, the absence of the receptor caused impairment of mitochondrial integrity and, finally, cell death. Our data reveal that VDR plays a central role in protecting cells from excessive respiration and production of ROS that leads to cell damage. Because we confirmed our observations in different models of both normal and cancer cells, we conclude that VDR is essential for the health of human tissues.

Autologous epidermal cell cultures (CEA) represent a possibility to treat extensive burn lesions, since they allow a significative surface expansion which cannot be achieved with other surgical techniques based on autologous grafting. Moreover currently available CEA preparations are difficult to handle and their take rate is unpredictable. This study aimed at producing and evaluating a new cutaneous biosubstitute made up of alloplastic acellular glycerolized dermis (AAGD) and CEA to overcome these difficulties. A procedure that maintained an intact basement membrane was developed, so as to promote adhesion and growth of CEA on AAGD. Keratinocytes were seeded onto AAGD and cultured up to 21 days. Viability tests and immunohistochemical analysis with specific markers were carried out at 7, 14, and 21 days, to evaluate keratinocyte adhesion, growth, and maturation. Our results support the hypothesis that this newly formed skin substitute could allow its permanent engraftment in clinical application.

Burn wounds give rise to the largest scars we can find in human pathology, influencing patients' quality of life. Despite the improved knowledge on pathophysiology, efficacy of the various treatments remains unsatisfactory. In this short review recent literature is examined with a focus on recent data on postburn pathological scars epidemiology and risk factors, which underline the high prevalence and the long evolution, pointing to identify this illness as a systemic inflammatory one, more frequent in women and in those of younger age, regulated by local factors relevant in wound healing.

Langerhans cells (LCs) are a specialized dendritic cell subset that resides in the epidermis and mucosal epithelia and is critical for the orchestration of skin immunity. Recent evidence suggest that LCs are involved in aberrant wound healing and in the development of hypertrophic scars and chronic wounds, which are characterized by a hypoxic environment. Understanding LCs biology under hypoxia may, thus, lead to the identification of novel pathogenetic mechanisms of wound repair disorders and open new therapeutic opportunities to improve wound healing. In this study, we characterize a previously unrecognized role for hypoxia in significantly affecting the phenotype and functional properties of human monocyte-derived LCs, impairing their ability to stimulate naive T cell responses, and identify the triggering receptor expressed on myeloid (TREM)-1, a member of the Ig immunoregulatory receptor family, as a new hypoxia-inducible gene in LCs and an activator of their proinflammatory and Th1-polarizing functions in a hypoxic environment. Furthermore, we provide the first evidence of TREM-1 expression in vivo in LCs infiltrating hypoxic areas of active hypertrophic scars and decubitous ulcers, pointing to a potential pathogenic role of this molecule in wound repair disorders.Hypoxia modulates surface molecule expression and cytokine profile in Langerhans cells. Hypoxia impairs human Langerhans cell stimulatory activity on naive T cells. Hypoxia selectively induces TREM-1 expression in human Langerhans cells. TREM-1 engagement stimulates Langerhans cell inflammatory and Th1-polarizing activity. TREM-1 is expressed in vivo in Langerhans cells infiltrating hypoxic skin lesions.

The aim of this work was to evaluate differences in energy flows between normal and immortalized cells when these distinct biological systems are exposed to environmental stimulation. These differences were considered using a constructal thermodynamic approach and were subsequently verified experimentally. The application of constructal law to cell analysis led to the conclusion that temperature differences between cells with distinct behaviour can be amplified by interaction between cells and external fields. Experimental validation of the principle was carried out on two cellular models exposed to electromagnetic fields. By infrared thermography we were able to assess small changes in heat dissipation measured as a variation in cell internal energy. The experimental data thus obtained are in agreement with the theoretical calculation, because they show a different thermal dispersion pattern when normal and immortalized cells are exposed to electromagnetic fields. By using two methods that support and validate each other, we have demonstrated that the cell/environment interaction can be exploited to enhance cell behavior differences, in particular heat dissipation. We propose infrared thermography as a technique effective in discriminating distinct patterns of thermal dispersion and therefore able to distinguish a normal phenotype from a transformed one.

Even in cells that are resistant to the differentiating effects of vitamin D, the activated vitamin D receptor (VDR) can downregulate the mitochondrial respiratory chain and sustain cell growth through enhancing the activity of biosynthetic pathways. The aim of this study was to investigate whether vitamin D is effective also in modulating mitochondria and biosynthetic metabolism of differentiating cells. We compared the effect of vitamin D on two cellular models: the primary human keratinocytes, differentiating and sensitive to the genomic action of VDR, and the human keratinocyte cell line HaCaT, characterized by a rapid growth and resistance to vitamin D. We analysed the nuclear translocation and features of VDR, the effects of vitamin D on mitochondrial transcription and the consequences on lipid biosynthetic fate. We found that the negative modulation of respiratory chain is a general mechanism of action of vitamin D, but at high doses, the HaCaT cells became resistant to mitochondrial effects by upregulating the catabolic enzyme CYP24 hydroxylase. In differentiating keratinocytes, vitamin D treatment promoted intracellular lipid deposition, likewise the inhibitor of respiratory chain stigmatellin, whereas in proliferating HaCaT, this biosynthetic pathway was not inducible by the hormone. By linking the results on respiratory chain and lipid accumulation, we conclude that vitamin D, by suppressing respiratory chain transcription in all keratinocytes, is able to support both the proliferation and the specialized metabolism of differentiating cells. Through mitochondrial control, vitamin D can have an essential role in all the metabolic phenotypes occurring in healthy and diseased skin.

Pentraxin 3 (PTX3) is a fluid-phase pattern recognition molecule and a key component of the humoral arm of innate immunity. In four different models of tissue damage in mice, PTX3 deficiency was associated with increased fibrin deposition and persistence, and thicker clots, followed by increased collagen deposition, when compared with controls. Ptx3-deficient macrophages showed defective pericellular fibrinolysis in vitro. PTX3-bound fibrinogen/fibrin and plasminogen at acidic pH and increased plasmin-mediated fibrinolysis. The second exon-encoded N-terminal domain of PTX3 recapitulated the activity of the intact molecule. Thus, a prototypic component of humoral innate immunity, PTX3, plays a nonredundant role in the orchestration of tissue repair and remodeling. Tissue acidification resulting from metabolic adaptation during tissue repair sets PTX3 in a tissue remodeling and repair mode, suggesting that matrix and microbial recognition are common, ancestral features of the humoral arm of innate immunity.

The evolution of the cosmic ray primary composition in the energy range 10 6 –10 7 GeV (i.e. the “knee” region) is studied by means of the e.m. and muon data of the Extensive Air Shower EAS-TOP array (Campo Imperatore, National Gran Sasso Laboratories). The measurement is performed through: (a) the correlated muon number ( N μ ) and shower size ( N e ) spectra, and (b) the evolution of the average muon numbers and their distributions as a function of the shower size. From analysis (a) the dominance of helium primaries at the knee, and therefore the possibility that the knee itself is due to a break in their energy spectrum (at E k He =(3.5±0.3)×10 6 GeV) are deduced. Concerning analysis (b), the measurement accuracies allow the classification in terms of three mass groups: light (p,He), intermediate (CNO), and heavy (Fe). At primary energies E 0 ≈10 6 GeV the results are consistent with the extrapolations of the data from direct experiments. In the knee region the obtained evolution of the energy spectra leads to: (i) an average steep spectrum of the light mass group ( γ p,He >3.1), (ii) a spectrum of the intermediate mass group harder than the one of the light component ( γ CNO ≃2.75, possibly bending at E k CNO ≈(6–7)×10 6 GeV), (iii) a constant slope for the spectrum of the heavy primaries ( γ Fe ≃2.3–2.7) consistent with the direct measurements. In the investigated energy range, the average primary mass increases from 〈ln A 〉=1.6–1.9 at E 0 ≃1.5×10 6 GeV to 〈ln A 〉=2.8–3.1 at E 0 ≃1.5×10 7 GeV. The result supports the standard acceleration and propagation models of galactic cosmic rays that predict rigidity dependent cut-offs for the primary spectra of the different nuclei. The uncertainties connected to the hadronic interaction model (QGSJET in CORSIKA) used for the interpretation are discussed.

Cell viability assessment in allograft skin is an essential step to ensure a supply of good quality allograft skin for clinical repair of wounds. It is widely recognised that ‘take’ of allografts is strongly influenced grafted by tissue viability. The aim of this study was to set-up storage protocols that maintain high viability of the allograft after harvest, treatment and storage. In this study, the viability of post-mortem allografts (n=350) harvested from 35 different donors, was investigated using the MTT salt assay. The conditions of preparation and storage of the allograft included: 1. Fresh skin samples (about 12, 30, and 60 h after harvesting). 2. The same specimens (stored at 4 and 37 °C) tested for at least 1 month. 3. Samples after cryopreservation and thawing. 4. Thawed specimens tested daily for at least 6 days. Parallel histomorphological analysis performed, under each of these conditions, showed a correlation between changes in structure and changes in viability as measured by the MTT quantitative assay. The viability index (VI) of skin is expressed as the ratio between the optical density (O.D.) produced in the MTT assay by the skin sample and its weight in grams. The percentage viability index is the ratio of the VI of the fresh sample (considered as 100% viability) and the value of specimens from the same harvest batch after storage or cryopreservation. The results indicated that samples tested within 12–30 h from harvesting have an average viability index of about 75 with little variation. Samples tested within 60 h have an average viability index of 40, showing a viability decrease of about 50%. A protocol to treat skin within a maximum of 30 h was, therefore, set-up. The data suggested that skin stored at 37 °C, undergoes a viability increase during the first 2 days after harvesting. However, the viability under these conditions then decreased very quickly. After 6 days of preservation at this temperature the samples were no longer viable (PVI = 0). The tissue structure started to become damaged after 3 days. On the other hand, skin stored at 4 °C, showed a very slow viability decrease. After 15 days, viability was still almost 25% of the fresh sample. The tissue architecture showed no signs of damage under these conditions until day 7 from harvesting. MTT analysis was performed on the specimens cryopreserved with DMSO at 10%. These measurements were compared to viability assessment of the same fresh skin samples (considered as 100%) that were analysed within 30 h from harvesting. The average PVI of thawed skin was 54% of the fresh sample. This result demonstrates that the viability of cryopreserved skin is comparable to the viability of fresh skin stored at 4 °C for 4 days. The PVI of thawed skin samples decreased dramatically within 24 h, and had reached 0% within 6 days.

A search for transient point sources of ultra-high-energy (UHE) γ-rays has been performed, based on the correlation of two extensive air shower arrays, BAKSAN (North Caucasus, 1700 m a.s.l., BAKSAN Neutrino Observatory, Russia) and EAS-TOP (Campo Imperatore, 2005 a.s.l., Laboratori Nazionali del Gran Sasso, Italy), which are located at very similar latitudes (φ≈43° N), and separated in longitude by Δλ≈33.7°. The search has been conducted at primary energy Eγ⩾5×1013 eV on the time scale of a daily source transit over the sky, observable in the northern hemisphere (19° The stability of the single detectors has been studied and verified up to high significance level. No coincident excess, not compatible with the expectations from statistical fluctuations, has been observed. The obtained upper limits to the rate of transient events, nγ, at 90% c.l., are • concerning the sky survey, in the declination band corresponding to the zenith, n γ /(Ωt) /(yr sr) with Φγ(Eγ>5×1013 eV)>2.0×10−11 cm −2 s−1 and duration Δt • concerning the candidate sources, e.g., for Markarian 421: nγ/t 5×1013 eV)>1.3×10−11 cm −2 s−1 and Δt A coincident episode from Markarian 421, observed on 15 January, 1994, with expected chance imitation rate nch= 0.01 is discussed.

The break observed in the electron shower size power law spectrum of Extensive Air Showers (EAS) at corresponding primary energy E-0 similar to (3 - 5)10(15) eV ("knee") is studied in different EAS components (electromagnetic and muonic) and at different atmospheric depths. A consistent description is obtained. The interpretation of data in terms of primary composition, and following the most accepted high energy interaction models, leads to an increasing average primary mass in this energy range. The study of such behaviour is expected to provide a crucial information for the understanding of the physical parameters that characterize the break for the different primaries.

The cross section for large p t jet production for transverse momentum 10≤ p t ≤20 GeV/c and rapidity 1.6≤ η ≤2.6 in p – N ( p –`air') interactions is studied from the analysis of multicore Extensive Air Showers recorded in the EAS–TOP calorimeter. The projectiles are the leading particles interacting at atmospheric depths between 250 and 480 cm −2 , the CMS energy of interaction being s ≈500 GeV. The slope of the p t distribution agrees with the expected one from the p – p collider data at the same CMS energy. The measured jet production cross section in p – N interactions with respect to p – p interactions is (dσ/dp t ) pN jet =(dσ/dp t ) p p jet ·A α with α =1.56±0.07 for A =14.7 (average mass number of `air' nuclei). Such value is compatible within the experimental uncertainties with the one obtained in p –nucleus accelerator measurements at s ≃30 GeV in the same range of transverse momentum and rapidity. No indication of increase of α with energy, i.e. of the entity of the `Cronin effect', is found.

Hypertrophic scarring is a skin disorder that occurs after wounding and thermal injury. There is accumulating evidence that immunologic processes such as infiltration of activated T lymphocytes and altered cytokine production may play a role in the formation of hypertrophic scars. Interleukin-15, a cytokine identified as a T cell growth factor, also acts as a chemoattractant for T cells and has pro-inflammatory properties. We investigated the expression and the role of this cytokine in hypertrophic scarring. IL-15 expression was compared in skin biopsies of hypertrophic scars (HS) both in active (AHS) and in remission (RHS) phases, in normotrophic scars (NTS) and in normal skin using reverse transcriptase-polymerase chain reaction and immunohistochemistry. IL-15 expression in HS was significantly higher than in NTS or normal skin. Furthermore, AHS expressed higher levels of IL-15 than RHS. Immunohistologic analysis of AHS samples showed strong IL-15 immunoreactivity in keratinocytes and Langerhans cells in the epidermis and in macrophages, fibroblasts, and dermal dendritic cells in the dermis. High levels of IL-15 expression in AHS correlated with abundant infiltration of activated CD3+ cells. Ex vivo experiments indicate that IL-15 can sustain the proliferative response of T cells derived from AHS but not from RHS and NTS. In addition, IL-15 prevents both cytokine deprivation and activation-induced apoptosis of T cells derived from AHS. Taken together, these results suggest that IL-15 can be involved in the recruitment, proliferation, and apoptosis inhibition of T cells in AHS. The findings that the evolution from an AHS to a RHS is associated with a decrease in IL15 expression, and with a loss of IL-15 responsiveness in ex vivo-cultured T cells, indicate that this cytokine plays an important role in the biology of pathologic scar formation.

The cytokine profile of T cell clones (TCC) from the dermis and epidermis of burn patients with hypertrophic scars (HS) in active (AHS) and remission phases (RHS) was determined in this study. We found that AHS tissues are heavily infiltrated by Type 0-Type 1 polarized CD3+ lymphocytes producing high IFN-gamma and low IL-4 levels. Analysis of their surface marker phenotype showed that the high IFN-gamma production was shared equally between the CD4+ TCRalpha/beta and CD8+ TCRalpha/beta clones. The profile of TCC from RHS tissues revealed pronounced infiltration of Type 0-Type 1 polarized lymphocytes with an even more evident Type 1 profile. However, the levels of IFN-gamma produced by RHS-derived TCC were 4-6 times lower than those produced by AHS-derived TCC. These data show that high levels of IFN-gamma produced by Type 0-Type 1 lymphocytes infiltrating HS are a feature of AHS, whereas reduction of this ability to produce high levels of IFN-gamma, though without a shift towards a Type 0-Type 2 phenotype through an increase in IL-4, is characteristic of RHS.

The amplitude and phase of the first harmonic of the sidereal anisotropy of cosmic rays at E 0 = 2·10 14 eV have been measured by the EAS-TOP experiment (Campo Imperatore, 2005 m a.s.l., LNGS, Italy) and are respectively (at the equatorial plane): A sid = (3.73±0.57)·10 −4 ; φ sid = 1.82±0.49 h lst, with significance 6.5 s.d. The result is supported by different consistency checks. From the comparison with lower energy data, it follows that the amplitude and the phase of the cosmic ray anisotropy are constant over the energy range 10 12 – 2·10 14 eV. This implies at these energies a constant influence of the local interstellar medium on the propagation of cosmic rays.

The cosmic ray energy spectrum in the range E-o = 10(15)-10(16) eV (including the region of the steepening, "knee") is studied by means of the EAS-TOP array (Campo Imperatore, Gran Sasso Laboratories, atmospheric depth 820 g cm(-2)). Measurements of the electromagnetic size (N-e = total number of charged particles at the observation level) are performed as a function of zenith angle with statistical accuracies of a few percent. The change of slope of the spectrum is observed in each bin of zenith angle at size values decreasing with increasing atmospheric depth. Its attenuation is compatible with the one of shower particles (Lambda(e) = 219 +/- 3 g cm(-2)). This observation provides a consistency check, supporting a normal behaviour of showers at the break, that make plausible astrophysical interpretations based on an effect on primaries occurring at a given primary energy. The break has a "sharp" shape (i.e., within experimental errors is compatible with two intersecting power laws) that represents a constraint with which any interpretation has to match. The change of slope of the power law index reproducing the size spectrum is Delta gamma = 0.40 +/- 0.09. The derived all particle energy spectrum is in good agreement with the extrapolation of the direct measurements at low energies and with other EAS data at and above the knee. Power laws fits to the energy spectrum below and above the knee give tin units of m(-2) s(-1) sr(-1) TeV-1) S(E-o) = (3.48 +/- 0.06) x 10(-10) (E-o/2300)(-2.76+/-0.03) for 900 TeV < E-o < 2300 TeV and S(E-o) = (3.77 +/- 0.08) x 10(-11) (E-o/5000)(-3.19+/-0.06) for 5000 TeV < E-o < 10(4) TeV. The systematic uncertainties connected to the interaction model and the primary composition are discussed. (C) 1999 Elsevier Science B.V.

It has been suggested that immunological factors play a major role in the pathogenesis of hypertrophic scars. Characteristically there is an onset of typical clinical features and, after a variable period of activity, there is a phase of remission. The factors which produce this progressive change in the scars are not clear, and it is not known if their manipulation could provide a therapeutic progress. In order to further investigate the pathophysiology, morphological studies have been performed. In active hypertrophic scars lymphocytic infiltrates are abundant. Among them activated T-cells represent 70% of infiltrates while in normotrophic scar activated T-cells make up 30% of lymphocytes, suggesting a role of these cells in the mechanisms leading to scar hypertrophy. Upon activation, the lesional T-cells release several cytokines which may induce anomalous expression of activation markers (HLA-DR, ICAM-1, CD36, IL-2R) on keratinocytes and fibroblasts of hypertrophic tissue. A wide range of cytokines has been examined: among those analyzed the only one that changes in the remission phase is IFNγ. In fact, IFNγ is highly expressed in lymphocytes in active hypertrophic scars while it is less expressed in the remission phase and in control samples.

In this study, skin-infiltrating cells were characterized in both the active and remission phases of post-burn hypertrophic scar biopsies. Immunohistochemistry examination of active phase samples showed an abundant presence of Langerhans cells, T cells, macrophages, a low presence of natural killer cells and the lack of B lymphocytes. In active hypertrophic scars T lymphocytes infiltrate deep into the superficial dermis and are also observed in the epidermis: CD3+ cells were present at about 222 +/- 107 per 0.25 mm2. In particular the analysis of lymphocyte subpopulations showed that CD4+ T cells predominate in the dermis as well as in the epidermis of active hypertrophic scars whereas CD8+ cells were less well represented (CD4/CD8 ratio is 2.06). This distribution was also shown in remission phase samples and in normotrophic scar specimens, although the lymphocyte number was significantly lower. Approximately 70 per cent of T lymphocytes present in the tissue involved in active phase hypertrophic scar samples were activated (positive with anti-HLA-DR and IL-2 receptor antibodies) which is significantly higher than remission phase hypertrophic and normotrophic scars, in which positivity was 40 and 38 per cent, respectively. Upon activation, the lesional lymphocytes release several cytokines, locally and transiently, that interact with specific receptors in response to different stimulation. Central to the immune hypothesis of hypertrophic scars is that some of the T-cell lymphokines act on keratinocytes, fibroblasts and other cell types to induce changes characteristic of these scars. The presence and close proximity of activated T lymphocytes and antigen-presenting cells of various phenotypes in both the epidermis and dermis of hypertrophic tissues provides strong circumstantial evidence of a local immune response. However, the manner in which T cells achieve and maintain their activated state in hypertrophic tissues is not yet known, and both antigen-dependent and independent mechanisms may contribute.

The importance of an adequate CNGS beam monitor at the Gran Sasso Laboratory has been stressed in many papers. Since the number of internal $\nu_\mu$ CC and NC interactions in the various detectors will not allow to collect statistics rapidly, one should also be able to detect the $\nu_\mu$ CC interactions in the upstream rock. In this study we have investigated the performances of the LVD detector as a monitor for the CNGS neutrino beam. Thanks to its wide area ($13 \times 11 m^2$ orthogonal to the beam direction) LVD can detect about 120 muons per day originated by $\nu_\mu$ CC interactions in the rock. The LVD total mass is $\sim2 kt$. This allows to get 30 more CNGS events per day as internal $(NC + CC)$ $\nu_\mu$ interactions, for a total of $\sim 150$ events per day. A 3% statistical error can be reached in 7 days. Taking into account the time characteristics of the CNGS beam, the cosmic muon background can be reduced to a negligible level, of the order of 1.5 events per day., Comment: 15 pages, 11 figures, submitted to Nucl. Instr. Meth. Phys. Res. A

The mechanical behaviour of skin is very important as regards dermatology, surgery and impact trauma. Engineered skin substitutes can bring significant medical benefit, in particular to patients with extensive burn wounds, even if current skin substitutes do not restore normal skin anatomy and its natural mechanical properties. This work considers the mechanical characterization of a particular layer of skin: dermis. Dermis can be used as a filling material and as support in different areas of reconstructive plastic surgery such as post mastectomy reconstructive surgery and abdominal surgery. The aim was to verify the influence of the decellularization treatment on its properties. The specimens were subjected to uniaxial static tests performed with Bose Electroforce ® 3200 and experimental data were represented with engineering and real time stress-strain curves. To begin, descriptive parameters were identified for stress vs. strain curves, such as ultimate tensile strength and maximum Young’s modulus, and they were subsequently compared through multivariate analysis of variance to determine the influence of specimen cut orientation and decellularization treatment duration. Dermis, that had been decellularized over 5 or 6 weeks, exhibited mechanical properties comparable with natural ones and ultimate tensile strength and maximum Young’s modulus were shown to be considerably higher in real time curves than in engineering ones.

We present the results of a search for Gamma Ray Bursts at energies Eγ ≥ 10 GeV and Eγ ≥ 100 TeV made by the EAS-TOP Air Shower Array in correlation with ∼ 50 events detected by BATSE in the last 3 years.

Human Acellular Dermal Matrices (HADM) are employed in various reconstructive surgery procedures as scaffolds for autologous tissue regeneration. The aim of this project was to develop a new type of HADM for clinical use, composed of glycerolized reticular dermis decellularized through incubation and tilting in Dulbecco's Modified Eagle's Medium (DMEM). This manufacturing method was compared with a decellularization procedure already described in the literature, based on the use of sodium hydroxide (NaOH), on samples from 28 donors. Cell viability was assessed using an MTT assay and microbiological monitoring was performed on all samples processed after each step. Two surgeons evaluated the biomechanical characteristics of grafts of increasing thickness. The effects of the different decellularization protocols were assessed by means of histological examination and immunohistochemistry, and residual DNA after decellularization was quantified using a real-time TaqMan MGB probe. Finally, we compared the results of DMEM based decellularization protocol on reticular dermis derived samples with the results of the same protocol applied on papillary dermis derived grafts. Our experimental results indicated that the use of glycerolized reticular dermis after 5 weeks of treatment with DMEM results in an HADM with good handling and biocompatibility properties.

Extensive Air Showers at large zenith angles θ > 70° (Horizontal Air Showers, HAS) are observed at the EAS-TOP array at Campo Imperatore (Gran Sasso Laboratories). The rate of these events exceeds the one due to primary cosmic rays (at this angles) and therefore these showers have to be generated by penetrating particles. Assuming that they are produced by atmospheric muons we derived the muon flux as Fμ(> 30 TeV) = 1.1 × 10−11cm−2s−1sr−1, in good agreement with the underground measurements. The upper limits for diffuse neutrino radiation from these measurements is Iν(> 105GeV) dI ν e (E 0 )/dE ν e −18 cm −2 s −1 sr −1 GeV −1 , for the resonant ( E 0 = m W 2 2m e = 6.4 × 10 6 GeV ) neutrinos.

The present study shows that the decrease of TNF alpha in postburn hypertrophic scars is due to a decrease in the steady-state level of TNF alpha mRNA and thus to an altered biosynthesis of the cytokine. Thirteen scars, including seven hypertrophic and six normotrophic scars, were tested for TNF alpha mRNA production by a semiquantitative reverse polymerase chain reaction (PCR) method. TNF beta and beta actin were tested as a control. Six out of six normotrophic scar samples amplified with primers for TNF alpha showed a positive PCR signal up to the 1:32 dilution. On the contrary all the hypertrophic tested samples (7/7) had a positive PCR signal only at the 1:1 or 1:2 dilution. All samples, both normotrophic and hypertrophic, were homogeneous as to TNF beta production.

Genetic experiments established that p63 is crucial for the development and maintenance of pluri-stratified epithelia and KLF4 for the barrier function of the skin. KLF4 is one of the factors that reprogram differentiated cells to iPS. We investigated the relationship between p63 and KLF4 using RNA interference, overexpression, chromatin immunoprecipitation and transient transfections with reporter constructs. We find that p63 directly represses KLF4 in normal keratinocytes (KCs) by binding to upstream promoter sites. Unlike p63, KLF4 levels are high in the upper layers of human skin and increase upon differentiation of KCs in vitro. In HaCaT KCs, which harbor two mutant alleles of p53, inactivation of p63 and of mutant p53 leads to KLF4 repression. p63 and p53 mutants are bound to sites in the KLF4 core promoter. Importantly, expression of the H179Y and R282Q p53 mutants in primary KCs is sufficient to activate endogenous KLF4. Finally, immunohistochemical analysis of tissue arrays confirms increased coexpression of KLF4 and mutant p53 in squamous cell carcinomas. Our data indicate that suppression of KLF4 is part of the growth-promoting strategy of p63 in the lower layers of normal epidermis, and that tumor-predisposing p53 mutations hijack p63 to a different location on the promoter, turning it into an activator of this reprogramming factor.

UHE cosmic rays are studied by means of the detectors of the different components of secondaries produced by their interactions in the atmosphere (EAS). We describe and discuss the reconstruction techniques and accuracies of the e.m. detector of EAS-TOP. They allow, besides independent high resolution measurements of UHE γ-ray astronomy, good correlation possibilities with the detectors of the different EAS components.