Long-term safety and efficacy of lentiviral hematopoietic stem/progenitor cell gene therapy for Wiskott-Aldrich syndrome.

Patients with Wiskott-Aldrich syndrome (WAS) lacking a human leukocyte antigen-matched donor may benefit from gene therapy through the provision of gene-corrected, autologous hematopoietic stem/progenitor cells. Here, we present comprehensive, long-term follow-up results (median follow-up, 7.6 years) (phase I/II trial no. NCT02333760 ) for eight patients with WAS having undergone phase I/II lentiviral vector-based gene therapy trials (nos. NCT01347346 and NCT01347242 ), with a focus on thrombocytopenia and autoimmunity. Primary outcomes of the long-term study were to establish clinical and biological safety, efficacy and tolerability by evaluating the incidence and type of serious adverse events and clinical status and biological parameters including lentiviral genomic integration sites in different cell subpopulations from 3 years to 15 years after gene therapy. Secondary outcomes included monitoring the need for additional treatment and T cell repertoire diversity. An interim analysis shows that the study meets the primary outcome criteria tested given that the gene-corrected cells engrafted stably, and no serious treatment-associated adverse events occurred. Overall, severe infections and eczema resolved. Autoimmune disorders and bleeding episodes were significantly less frequent, despite only partial correction of the platelet compartment. The results suggest that lentiviral gene therapy provides sustained clinical benefits for patients with WAS.

Piezoelectric displacement mapping of compliant surfaces by constant-excitation frequency-modulation piezoresponse force microscopy.

A simple experimental method for piezoresponse force microscopy (PFM) measurements for reliable evaluation of piezoelectric surface displacements even on compliant surfaces is proposed based on atomic force microscopy (AFM) operated in frequency-modulation (FM) dynamic mode with constant excitation (CE), by using non-contact mode cantilevers. Surface displacement by piezoelectric effect after application of an electric potential to the conductive AFM probe translates into a likewise variation of the probe oscillation amplitude, while the related electrostatic forces mainly affect the oscillator resonant frequency, and cantilever bending is limited due to their high stiffness. Our non-contact CE-FM-PFM method is shown to reduce electrostatic force contributions as compared to contact-PFM modes. Converse piezoelectric effect mapping is demonstrated on poly(vinylidenefluoride) nanofibers obtained by electrospinning.

Rapid Maxillary Expander and Eruption Guidance Appliance therapy in skeletal Class II: cephalometric considerations.

AIM: The use of rapid maxillary expander (RME) combined with eruption guidance appliances (EGA) represents a valid method in the treatment of skeletal Class II deep bite patients. In this paper a retrospective cephalometric study of the effects of RME and EGA therapy in Class II deep bite patients is described and compared with the treatment effects of patients with the same malocclusion treated only by RME. MATERIALS AND METHODS: Pre and post treatment cephalometric radiographs of 47 Class II division 1 deep bite growing patients treated with RME followed by EGA (mean age 9.65 years) (Group 1) were compared with those obtained from a control group (Group 2) of 44 patients (mean age 9.34 years) treated with RME only. The following cephalometric values were analized: SNA; SNB; ANB; SN-PNS.ANS; SN-Go.GN; N-Me; S-Go. RESULTS: Improvement in maxillo-mandibular relationship was observed in both groups with significantly higher decrease of SNA and ANB in group 1. The cranio-maxillary relation shows a tendency to grow downward and backward in both groups. Both anterior and posterior total facial heights showed a significant increase in group 1. CONCLUSIONS: RME followed by EGA therapy in Class II skeletal deep bite patients has shown better results compared with RME therapy without functional appliance.

Assessment of climate change effects on mountain ecosystems through a cross-site analysis in the Alps and Apennines.

Mountain ecosystems are sensitive and reliable indicators of climate change. Long-term studies may be extremely useful in assessing the responses of high-elevation ecosystems to climate change and other anthropogenic drivers from a broad ecological perspective. Mountain research sites within the LTER (Long-Term Ecological Research) network are representative of various types of ecosystems and span a wide bioclimatic and elevational range. Here, we present a synthesis and a review of the main results from ecological studies in mountain ecosystems at 20 LTER sites in Italy, Switzerland and Austria covering in most cases more than two decades of observations. We analyzed a set of key climate parameters, such as temperature and snow cover duration, in relation to vascular plant species composition, plant traits, abundance patterns, pedoclimate, nutrient dynamics in soils and water, phenology and composition of freshwater biota. The overall results highlight the rapid response of mountain ecosystems to climate change, with site-specific characteristics and rates. As temperatures increased, vegetation cover in alpine and subalpine summits increased as well. Years with limited snow cover duration caused an increase in soil temperature and microbial biomass during the growing season. Effects on freshwater ecosystems were also observed, in terms of increases in solutes, decreases in nitrates and changes in plankton phenology and benthos communities. This work highlights the importance of comparing and integrating long-term ecological data collected in different ecosystems for a more comprehensive overview of the ecological effects of climate change. Nevertheless, there is a need for (i) adopting co-located monitoring site networks to improve our ability to obtain sound results from cross-site analysis, (ii) carrying out further studies, in particular short-term analyses with fine spatial and temporal resolutions to improve our understanding of responses to extreme events, and (iii) increasing comparability and standardizing protocols across networks to distinguish local patterns from global patterns.

Continuous multilayered composite hydrogel as osteochondral substitute.

Cartilage is a highly organized avascular soft tissue that assembles from nano-to macro-scale to produce a complex structural network. To mimic cartilage tissue, we developed a stable multilayered composite material, characterized by a tailored gradient of mechanical properties. The optimized procedure implies chemical crosslinking of each layer directly onto the previous one and ensures a drastic reduction of the material discontinuities and brittleness. The multilayered composite was characterized by infrared spectroscopy, differential scanning calorimetry, thermogravimetry, and scanning electron microscopy in order to compare its physico-chemical characteristics with those of cartilage tissue. The rheological behavior of the multilayered composite was similar to that of human cartilage. Finally its cytocompatibility toward chondrocytes and osteoblasts was evaluated.

A moiré deflectometer for antimatter.

The precise measurement of forces is one way to obtain deep insight into the fundamental interactions present in nature. In the context of neutral antimatter, the gravitational interaction is of high interest, potentially revealing new forces that violate the weak equivalence principle. Here we report on a successful extension of a tool from atom optics--the moiré deflectometer--for a measurement of the acceleration of slow antiprotons. The setup consists of two identical transmission gratings and a spatially resolving emulsion detector for antiproton annihilations. Absolute referencing of the observed antimatter pattern with a photon pattern experiencing no deflection allows the direct inference of forces present. The concept is also straightforwardly applicable to antihydrogen measurements as pursued by the AEgIS collaboration. The combination of these very different techniques from high energy and atomic physics opens a very promising route to the direct detection of the gravitational acceleration of neutral antimatter.

Synthesis of esters of androgens with unsaturated fatty acids for androgen requiring therapy.

Androgens' metabolism and activity are gaining a more and more important role in human physiology particularly referring to aging and to neurodegenerative diseases. Androgen treatment is often required for long-lasting disorders. In order to improve their duration and effects, androgens can be administered as esters of carboxylic acids. The novelty of our research is the use of esters of androgens with specific unsaturated fatty acids, in order to reduce possible side effects particularly related to chronic pathologies with altered lipid homeostasis such as X-linked adrenoleukodystrophy and cardiovascular disorders. Thus the esters of the main androgenic substances testosterone, dihydrotestosterone (DHT) and their metabolite 5α-androstan-3α,17ß-diol were chemically obtained by coupling with different unsaturated fatty acids. To this aim, fatty acids with various degree of unsaturation and belonging to different series were selected. Specifically, oleic acid (18:1, n-9), linoleic acid (18:2, n-6), and the n-3 fatty acids, α-linolenic acid (18:3), eicosapentaenoic acid (EPA, 20:5), and docosahexaenoic acid (DHA, 22:6) were used obtaining corresponding esters with acceptable yields and good degree of purity. All the synthesized compounds were tested for their cytotoxic activities in mouse NIH3T3 and human astrocyte cell lines. The esters demonstrated good tolerability and no in vitro cytotoxic effect in both cell cultures. After these promising preliminary results, the esters will be suitable for in vivo studies in order to ascertain their pharmacokinetic characteristics and their biological effects.

High resolution self-mixing laser rangefinder.

This work describes the development of a novel laser instrument for non-contact absolute distance measurements, working in safety class I. The physical principle of operation is based on the self-mixing effect in a laser diode: the laser diode wavelength is modulated through a change of the supply current, inducing a self-mixing signal. The frequency of the produced fringes depends on the target distance, therefore a particular algorithm is applied, in real-time, to extract the frequency tone of the fringes, even in the case of very low signal-to-noise ratio. The final low-cost instrument is realized with an embedded digital signal processor and works in real-time over diffusive targets, with resolution better than 100 µm for distances up to 2 m.

New perspectives in cell communication: Bioelectromagnetic interactions.

This paper explores physical signalling in biological communications, the so-called biophysical pathways, and especially the role of electromagnetic signalling in cell-cell interactions. The experiments were designed to evaluate whether different cell populations physically interfere when incubated in separate Petri dishes placed in close proximity. Two different cell populations, immortalized mouse fibroblasts (NIH3T3) and adult human microvascular endothelial cells (HMVECad) were selected and seeded in separate polystyrene Petri dishes. Dishes seeded with NIH3T3 were then placed on top of those seeded with HMVECad at distances of 4mm and 11mm. A black filter was placed between dishes containing the two cell populations in another experiment, to prevent transmission of electromagnetic radiation between the two. Cell number and morphology of NIH3T3 and endothelial cells were found to be modified in dishes without the black filter, suggesting that specific signals emitted by the cells were transmitted through the polystyrene wall, affecting cell proliferation rate and morphology, even though the cells were growing in separate dishes.

Reelin levels are increased in synovial fluid of patients with rheumatoid arthritis.

OBJECTIVES: To evaluate the presence and the glycosylation pattern of reelin in synovial fluid and serum of patients affected by different rheumatic pathologies. METHODS: Reelin levels were evaluated in patients affected by rheumatoid arthritis (RA), psoriatic arthritis (PsA), spondyloarthritis (SpA) and osteoarthritis (OA). Reelin semi-quantitative assays were performed by western blot. The glycosylation pattern was evaluated by immunoblotting performed by sepharose conjugated lectins. RT-PCR was used to detect the presence of mRNA encoding for reelin and its receptors. RESULTS: Reelin is detectable in both sinovial fluids and sera and its levels are more elevated in patients affected by RA with respect to those affected by other inflammatory and non inflammatory joint diseases. The glycosylation pattern of the protein differs in synovial fluid and serum. Fibroblast-like synoviocytes (FLS) express the mRNAs encoding for reelin and its receptors. CONCLUSIONS: Since its levels are higher in RA then in the other analysed pathologies, reelin can represent a candidate suitable for the differential diagnosis of this pathology. Moreover, the observation that this protein is encoded by FLS and differentially glycosylated in blood and synovial fluid supports the hypothesis that it is locally produced in the joints, where it could play an important role in RA development and maintenance.

Impact of involvement of individual joint groups on subdimensions of functional ability scales in juvenile idiopathic arthritis.

OBJECTIVE: To investigate the influence of arthritis in individual joint groups on subdimensions of functional ability questionnaires in children with juvenile idiopathic arthritis (JIA). METHODS: 206 patients were included who had the Childhood Health Assessment Questionnaire (C-HAQ) and the Juvenile Arthritis Functionality Scale (JAFS) completed simultaneously by a parent and received a detailed joint assessment. In each patient, joint involvement (defined as presence of swelling, pain on motion/tenderness and/or restricted motion) was classified in 3 topographic patterns: Pattern 1 (hip, knee, ankle, subtalar and foot joints); Pattern 2 (wrist and hand joints); Pattern 3 (elbow, shoulder, cervical spine and temporomandibular joints). Frequency of reported disability in each instrument subdimension was evaluated for each joint pattern, present either isolatedly or in mixed form. RESULTS: Among patients with Pattern 1, the JAFS revealed the greatest ability to capture and discriminate functional limitation, whereas impairment in the C-HAQ was more diluted across several subdimensions. Both C-HAQ and JAFS appeared to be less reliable in detecting functional impairment in the hand and wrist (Pattern 2) than in other body areas. Overall, the JAFS revealed a superior ability to discriminate the relative functional impact of impairment in individual joint groups among patients with mixed joint patterns. CONCLUSION: In children with JIA, a functional measure focused to assess the function of individual joint groups (the JAFS) may detect with greater precision the functional impact of arthritis in specific body areas than does a standard questionnaire based on the assessment of activities of daily living (the C-HAQ).

The magnitude of early response to methotrexate therapy predicts long-term outcome of patients with juvenile idiopathic arthritis.

OBJECTIVE: To investigate the relationship between the magnitude of clinical response in the first 6 months of methotrexate (MTX) therapy and long-term outcome in children with juvenile idiopathic arthritis (JIA). METHODS: The clinical charts of 125 JIA patients who were started with MTX and then followed for at least 5 years were reviewed. Based on the level of American College of Rheumatology (ACR) Pediatric response at 6 months, patients were divided in four mutually exclusive groups: (1) non-responders, (2) responders at 30%, (3) responders at 50%, and (4) responders at 70%. The long-term outcome in each response group was evaluated by calculating the percentage change in active and restricted joint counts from baseline to 1, 2 and 5 years and the frequency of inactive disease at 5 years. RESULTS: At 6 months, 42 patients were classified as non-responders, 24 as 30% responders, 26 as 50% responders, and 33 as 70% responders. Patients who had achieved a 70% response showed a significantly greater percentage improvement in active joint count between baseline to 5 years compared with non-responders and 30% responders, and a significantly greater percentage improvement in restricted joint count between baseline to 5 years compared with 30% responders. The 70% responders also had a greater frequency of inactive disease at 5 years compared with 30% responders, CONCLUSIONS: Our results show that the achievement of an ACR Pediatric 70 response at 6 months after start of MTX therapy predicts a more favorable long-term outcome of patients with JIA.

Fibroblast cell behavior on bound and adsorbed fibronectin onto hyaluronan and sulfated hyaluronan substrates.

The effect of fibronectin protein (Fn) coating onto polysaccharide layers of hyaluronic acid (Hyal) and its sulfated derivative (HyalS) on fibroblast cell adhesion was analyzed. The Hyal or HyalS were coated and grafted on the glass substrate by a photolithographic method. The Fn coating was achieved by two different routes: the immobilization of Fn by covalent bond to the polysaccharide layers and the simple adsorption of Fn onto Hyal and HyalS surfaces. AFM, SEM, and ATR-FTIR techniques were used for the chemical and topographical characterization of the surfaces. According to AFM and SEM data, the surface topography was dependent on the method used to cover the polysaccharide layers with the protein. ATR-FTIR analysis supplied information about the rearrangement of Fn after the interaction (adsorption or binding) with the Hyal and the HyalS. The conformational changes of the Fn were minimal when it was simply adsorbed on HyalS surfaces and larger once bound, whereas on the Hyal layer the protein underwent a bigger conformational change once adsorbed and covalently grafted. Then, the biological characterization was carried out by analyzing the human diploid skin fibroblasts adhesion on these surfaces. The morphology of fibroblasts was evaluated by SEM, whereas the dynamics of fibroblasts movement were recorded by a time-lapse system. Cell variations in area, perimeter, and length were analyzed at 2, 4, and 6 h. It was found that the addition of Fn (covalently bound or merely adsorbed) was fundamental in the promotion of fibroblasts adhesion and spreading. The greatest adhesion occurred onto HyalS layers covered by the adsorbed Fn.

Micropatterned hyaluronan surfaces promote lymphatic endothelial cell alignment and orient their growth.

The implant of a biocompatible device capable of guiding lymphatic vessel regeneration in patients who underwent removal of lymph nodes might contribute to restoring an efficient lymphatic drainage and help to prevent the occurrence of lymphedema. The aim of this study was to evaluate whether a microstructured surface could provide a guidance for the growth of cultured lymphatic endothelial cells. The presence of microstructures on a surface permits the control of cell adhesion, migration, proliferation, and differentiation. We report here that lymphatic endothelial cells align on microstructures of alternating hyaluronan and aminosylanized glass stripes obtained by photoimmobilization. Cells consistently spread and proliferate only on aminosylanized glass. They orient parallel to the longitudinal axis of the stripe. A pattern of alternating stripes of aminosylanized glass uniformly covered by elongated cells and of hyaluronan devoid of cells eventuallyforms. The presence of alpha(v)-integrins along cell borders of cells in search of contact with each other and at the leading edge of migrating cells, sites where new focal adhesions are presumably formed, indicates that integrin-mediated adhesion to the substrate guides cell migration along the microstructure. Micropatterned surfaces of hyaluronan thus proved to adequately orient the growth of cells allowing the regeneration of lymphatic endothelium in the desired direction.

The use of hyaluronan and its sulphated derivative patterned with micrometric scale on glass substrate in melanocyte cell behaviour.

Surface microfabrication techniques were widely utilised for the spatial control of in vitro cell behaviour. A photo-immobilisation procedure was utilised to create micropatterned surfaces: four different stripe patterns (100, 50, 25 and 10 microm) of hyaluronan (Hyal) and its sulphated derivative (HyalS) on silanised glass substrate were obtained.The morphological analysis showed that the surface topography showed regular stripes of 100, 50, 25 and 10 microm wide and ranging from 300 nm up to 1 microm in thickness. They reproduced the exact photo-mask pattern: glass stripes alternating with polysaccharide ones. On the contrary, Hyal microstructures showed just a topographic pattern as the glass stripes appeared to be covered by a thin layer of the macromolecule by TOF-SIMS. Cell adhesion studies demonstrated that melanocytes adhered and oriented within the first 2h of culture on HyalS microdomains and not on Hyal microstructures where they spread on glass substrate around the patterned area. Double photo-immobilised samples characterised by a 100 microm stripe pattern of Hyal or HyalS on the top of a continuous layer of the two polysaccharides were also created in order to investigate the effect of the topography on cell behaviour. The obtained results demonstrated that melanocytes adhered on HyalS stripes while on the Hyal micropatterned surfaces they spread on silanised glass substrate around the structured area, resulting in the exclusion of the topographic pattern.

Effect of subclinical hypothyroidism and obesity on whole-body and regional bone mineral content.

OBJECTIVE: The present investigation was aimed to evaluate the effect of subclinical hypothyroidism and obesity on bone mineral content (BMC) in different body segments. METHODS: Thirty-two premenopausal women (age: 37 +/- 9.9 years), with a wide range in body mass index (BMI), were studied. Subclinical hypothyroidism was defined by a basal TSH > or = 4 microU/l and/or a TRH-stimulated peak > or = 30 microU/l. For each subject, weight, height, BMI (weight/height(2)) and the waist/hip ratio were measured. Total BMC, total bone mineral density (BMD), leg BMC, leg BMD, trunk BMC, trunk BMD, arm BMC and arm BMD were determined using dual-energy X-ray absorptiometry. Thyroid function (basal and TRH-stimulated TSH, free T(3) and free T(4)) were determined from fasting blood samples for all subjects. RESULTS: Anova was conducted within all the groups to observe the effect of thyroid status and/or obesity on BMC and BMD. There was no statistical difference for age. Total BMC was affected by obesity (p < 0.05) but not by thyroid status, BMD of the legs was significantly influenced both by thyroid function and obesity (p < 0.01); total BMD was affected by hypothyroid status (p < 0.05). A direct relationship between leg BMD and TSH was demonstrated. CONCLUSION: Subclinical thyroid hypofunction and obesity seem to affect BMD differently in the body segments. An influence of gravitational force seems necessary in order to make evident the effect of subclinical hypothyroidism on bone. A condition of subclinical hypothyroidism should be considered when evaluating subjects for osteoporosis, since a BMD measured at the femoral neck may induce underestimation of initial osteoporosis.

Solution structure of hyaluronic acid oligomers by experimental and theoretical NMR, and molecular dynamics simulation.

The conformational properties of hyaluronic acid (HA) oligomers in aqueous solution were investigated by combining high-resolution NMR experimental results, theoretical simulation of NMR two-dimensional (2D) spectra by Complete Relaxation Matrix Analysis (CORMA), and molecular dynamics calculations. New experimental findings recorded for the tetra- and hexasaccharides enabled the stiffness of the HA and its viscoelastic properties to be interpreted. In particular, rotating frame nuclear Overhauser effect spectroscopy spectra provided new information about the arrangement of the glycosidic linkage. From (13)C NMR relaxation the rotational correlation time (tau(c)) were determined. The tau(c) were employed in the calculation of geometrical constraints, by using the MARDIGRAS algorithm. Restrained simulated annealing and 1 ns of unrestrained molecular dynamic simulations were performed on the hexasaccharide in a box of 1215 water molecules. The beta(1 --> 3) and beta(1 --> 4) glycosidic links were found to be rigid. The lack of rotational degree of freedom is due to direct and/or water-mediated interresidue hydrogen bonding. Both single or tandem water bridges were found between carboxylate group and N-acetil group. The carboxylate group of glucuronic acid is not involved in a direct link with the amide group of N-acetyl glucosamine and this facilitated bonding between the residue and the water molecules.

Cu(II) and Zn(II) complexes with hyaluronic acid and its sulphated derivative. Effect on the motility of vascular endothelial cells.

With the aim of improving the compatibility of biomaterials to be used for the construction of cardiovascular prosthesis, we have designed bioactive macromolecules resulting from chemical modifications of hyaluronic acid (Hyal). The stability constants of Cu(II) and Zn(II) complexes with the sulphated derivative of hyaluronic acid (HyalS3.5) were evaluated. Two different complexes have been found for each metal ion, CuL, Cu(OH)2L and ZnL, Zn(OH)2L (L means the disaccharide unit of the ligands) in aqueous solution at 37 degrees C. The dihydroxo Cu(II) complex was present in high percentage at pH=7.4. On the contrary, the Zn(II) ion was present with a relatively low percentage of both complexes. The ability to stimulate endothelial cell adhesion and migration was evaluated for Hyal, HyalS3.5 and their complexes with Cu(II) and Zn(II) ions. The results revealed that Hyal and [Cu(OH)2HyalS3.5](4.5)- induced cell adhesion, while [ZnHyalS3.5](2.5)- and [Zn(OH)2HyalS3.5](4.5)- inhibited the process. The chemotactic activity of increasing concentrations of the above complexes was also evaluated, demonstrating that [Cu(OH)2HyalS3.5](4.5)- complex at 1 microM concentration was the most active in inducing cell migration. These results have been also strengthened by analysing adherent cell migration in agarose. In conclusion, sulphated hyaluronic acid coordinated to Cu(II) seems to be a promising matrix molecule for the construction of cardiovascular prosthesis.

Effect of subclinical hypothyroidism on body fluid compartments.

OBJECTIVE: The present study was aimed to assess the effects of subclinical hypothyroidism on body composition (BC). SUBJECTS: Thirty-one women (age: 37 +/- 9.9 years) with a wide range of body mass index (BMI) were studied. Subclinical hypothyroidism was defined by a basal TSH > or = 4 mU/L and/or TRH stimulated peak > or = 30 mU/L. MEASUREMENTS: For each subject, weight, height, BMI, multifrequency bioelectrical impedance spectroscopy (BIS) and D2O and NaBr dilution tests were performed to assessed total body water (TBW) and extracellular water (ECW). Thyroid function (basal and TRH stimulated TSH, free T3, and free T4) were determined from fasting blood samples for all subjects. Total body dual energy X-ray absorptiometry (DXA) were used to measure fat mass (FM) and lean mass (Lean). RESULTS: The results of BIS were compared with the TBW and ECW estimated by the dilution techniques on the same individuals. The correlation was R2 = 0.65 for impedance at 5 kHz and ECW by NaBr and R2 = 0.72 for impedance at 100 kHz and TBW by D2O. Intracellular water (ICW) was calculated as differences between TBW and ECW measured by dilution methods. Percent of ECW and ICW were related to BMI (ANOVA, p < 0.001). No difference in TBW, body water distribution and body composition related to thyroid function was demonstrated. CONCLUSIONS: In our patients affected with subclinical hypothyroidism, with or without obesity, only obesity appeared related to TBW, ECW and ICW; the subclinical hypothyroidism, on the contrary, had no effect on compartments of body fluids. Bioimpedance is a valid tool to assess body fluid distribution in subclinical hypothyroidism.