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Background Intestinal ultrasound (IUS) is accurate to assess endoscopic activity in ulcerative colitis (UC). The Milan ultrasound criteria (MUC) is a validated scoring system to assess and grade endoscopic activity in UC. The most accurate cutoff value for MUC was > 6.2 for endoscopic activity (defined as a Mayo endoscopic score, MES ≥ 2). The aim of this study was to assess the predictive value of IUS and MUC for treatment response in a longitudinal cohort, using colonoscopy (CS) as reference standard. Methods Consecutive active UC patients starting biologic therapy were included. All patients underwent CS, IUS, clinical and faecal calprotectin (FC) evaluations prior commencing a biological therapy, and within one year (mean 9.4 months). In addition, patients were evaluated by IUS, clinical and FC assessments at week 12. The primary objective was to evaluate whether ultrasound improvement (defined as MUC ≤ 6.2) at week 12 predicted endoscopic improvement at reassessment (defined as MES ≤ 1). Endoscopic remission was defined as MES = 0. Results Forty-nine patients were included (59% under infliximab, 29% under vedolizumab, 8% under adalimumab, 4% under ustekinumab). MUC and MES correlated at reassessment (r= 0.767, p < 0.001). Ultrasound improvement at week 12 was the only independent predictor for MES ≤ 1 and MES = 0 at reassessment (OR 5.80, p = 0.010; OR 10.41, p = 0.041; respectively). Ultrasound improvement at week 12 showed NPV of 96% for detecting MES = 0. A ≥ 2 reduction of the MUC score predicted MES=0 (area under the curve, AUC 0.816). MUC ≤ 4.3 was the most accurate cut-off value for MES = 0 (AUC 0.876). The responsiveness ratio of Guyatt for the MUC was 1.73 and the standardized effect size ratio was 1.6. Both these values > 0.8 indicate a large effect of responsiveness for the MUC. Conclusion MUC is highly accurate to monitor treatment response. Ultrasound improvement after the induction period may predict long-term endoscopic response. The MUC may be used both in clinical trials and routine practice.

Background CD is classically seen as a progressive disease leading to bowel damage and disability. Disability has been proposed by the Spirit-IOIBD consensus1 as an endpoint in disease modification trials. However, there is a paucity of data regarding disability in CD patients at diagnosis. Methods The “Crohn′s Disease Cohort Study” (CROCO) is an ongoing prospective cohort aiming to study the longitudinal evolution of bowel damage and disability following diagnosis of CD. All patients are included within 12 months following CD diagnosis and will be followed over 5 years with serial assessments of bowel damage and disability. At recruitment patients completed an IBD disability score questionnaire (DS),2 comprising several domains: mobility, self-care, major daily life activities, gastrointestinal-related problems, mental health and interaction with the environment. We here describe the baseline DS and its association with disease features, disease activity score (Harvey-Bradshaw Index), and biomarkers of disease activity, on the first patients recruited into CROCO. Results Ninety-four patients were recruited, of which 66 filled at least 80% of the questions in the DS questionnaire; of those 55% were male and median age at diagnosis was 30.6 [IQR 24.2;38.8]. The baseline visit occurred at a median time of 6 months [IQR 2.6;9.2] after CD diagnosis. Ileal involvement (L1/L3) was reported in 82% of patients; 68% had B1 phenotype, and 26% presented extra-intestinal manifestations. Overall, 32% of patients had been hospitalised, and 6% had a history of CD-related surgery. Regarding medication [from diagnosis to baseline], 55% received steroids, 56% immunosuppressants, and 65% Anti-TNF therapy. The median DS was 79 [IQR 67;108.2] (range: 49;198); only 2 patients (3%) patients presented DS>162 (the central value of the DS), indicating no or mild disability. No correlations were found between clinical or analytical biomarkers of disease activity at the time of recruitment (Table 1). Associations between disease features and DS are displayed in Table 2. A history of CD-related hospitalisation was associated with a significantly higher DS (103 [IQR 79;157] versus 76.5 [65.5;97], p=0.013). No other associations were observed. Conclusion In a cohort of newly diagnosed CD patients, disability was mostly mild and independent of disease activity, disease location or behaviour; around 1/3 required hospitalisation within the first year of diagnosis, which was associated with increased disability. These data add to the growing concept that newly diagnosed disease represents a window of opportunity for intervention.

Background Up to 15% of patients with ulcerative colitis (UC) do not respond to medical therapies and ultimately require colectomy for disease control. Baseline endoscopic severity and failure to achieve endoscopic healing, as defined by a Mayo Endoscopic Subscore (MES) ≤ 1, following therapy have been associated with an increased risk of colectomy. Intestinal ultrasound severity, as defined by a Milan ultrasound criteria (MUC) score > 6.2, has been associated with an increased risk of colectomy. The aim of this study is to evaluate and compare MES and MUC in predicting the need for colectomy in patients with UC. Methods This is a double-center prospective observational cohort study. All consecutive adult UC patients between January 2016 and January 2020 requiring colonoscopy received intestinal ultrasound within 20 ± 12 days in a blinded fashion. Colectomies were evaluated during the follow-up. Univariable and multivariable Cox regression analyses were used to identify variables independently associated with colectomy risk. ROC analysis was used to compare baseline MES and MUC scores' performances in predicting colectomy. Results A total of 141 patients were enrolled (Table 1). Overall 13 patients underwent colectomy during 256.41 person-years of observation time. At baseline, patients requiring colectomy had increased mean values of MUC as compared to patients not undergoing surgery (6.84 ± 2.49 vs 10 ± 1.9, p Conclusion Ultrasound severity, as assessed by the MUC score, is superior to endoscopic severity in predicting the need for colectomy in patients with UC. A baseline MUC score of < 7.72 may rule out colectomy risk in UC patients.

Background Intestinal ultrasound (IUS) is accurate and non-invasive to detect and monitor disease activity in Crohn’s disease (CD). The Bowel Ultrasound Score (BUSS= 0.75 × bowel wall thickness + 1.65 × presence (1) or absence (0) of bowel wall flow) demonstrated high accuracy in detecting therapy-related changes, and good correlation with the Simple Endoscopic Score for CD (SES-CD). The most accurate cutoff value for BUSS was 3.52 for endoscopic remission (SES-CD ≤ 2).This study aimed to prospectively evaluate ultrasound remission as a relevant treatment target. Methods Consecutive patients with active CD (SES-CD > 2) starting biologic treatments were included. Patients underwent colonoscopy and IUS at baseline and after 1 year of treatment. Clinical, biochemical and ultrasound assessments were additionaly performed at week 12. Primary outcome was to evaluate whether ultrasound remission at week 12 could predict endoscopic remission at week 54. Endoscopic remission was defined as SES-CD ≤ 2, ultrasound remission was defined by BUSS ≤ 3.52, clinical remission was defined by HBI (Harvey-Bradshaw Index) < 5, and biochemical remission was defined by normalization of C-reactive protein (< 5 mg/L) and fecal calprotectin (three different thresholds were assessed: < 250 µg/g, < 100 µg/g, < 50 µg/g). Results Ninety-three patients were included (8 under infliximab, 40 under adalimumab, 5 under vedolizumab and 40 under ustekinumab). Eighteen patients (19%) and 36 patients (39%) achieved endoscopic remission and ultrasound remission at week 54, respectively. CD patients with ultrasound remission at week 12 were more likely to achieve endoscopic remission (odds ratio [OR] 6.88 [2.14–22.04]; p = 0.001) and ultrasound remission (OR 4.26 [1.61–11.24]; p = 0.003) at week 54. Calprotectin value < 50 µg/g at week 12 predicted endoscopic remission at univariable (OR 3.14 [1.04–9.45]; p = 0.041), but not at multivariable analysis. No other clinical or biochemical variables at week 12 predicted endoscopic and/or ultrasound remission at week 54. Conclusion Ultrasound remission achieved at week 12 may predict long-term endoscopic remission and may be a strategic treatment target, both in clinical practice and in clinical trials.

Background Ulcerative Colitis (UC) is a chronic, idiopathic intestinal inflammatory disease whose diagnosis requires colonoscopy with biopsy. There is an unmet need for sensitive, and easy-to-detect biomarkers helping a rapid and non-invasive diagnosis, and suitable for following disease progression. Extracellular vesicles (EVs) have been proposed as promising carriers of biomarkers. Nevertheless, their plasma levels remain very low and thus difficult to detect. We explored Low-Intensity Pulsed Ultrasound (LIPUS) as a novel and safe approach to enhance the mucosal release of EVs in experimental models of UC. Methods LIPUS was applied at a frequency of 38 kHz and at an intensity of 150 mW/cm2 for 3 min, using devices dedicated to in vitro or in vivo studies. Primary intestinal fibroblasts, Human Intestinal microvasculature endothelial cells (HIMEC) and peripheral blood mononuclear cells (PBMC) were isolated from UC patients and healthy volunteers (n=6). Cell viability was tested in Caco-2 and all primary cells using MTT assay. Computer acoustic simulations, allowing a reliable control of the energy dose in the colon, were carried out using k-Wave software to translate in vivo the LIPUS. Acute and chronic colitis models were induced in C57BL/6N mice by administration of dextran sodium sulfate (DSS) respectively 2 and 2.5% ad libitum in their drinking water for seven days for the acute and three cycles of DDS for the chronic. Mice were monitored daily for body weight loss, bleeding and stool consistency. EVs were characterized in supernatants, plasma and mucosa at NanoSight and inflammatory mediators detected 1, 2 and 24 h after LIPUS by ELISA assay. Apoptosis and cell proliferation were evaluated by TUNEL and Ki67 stainings. Results After LIPUS, all cells appeared 100% viable and no pro-inflammatory changes were observed. In Caco-2 cells, fibroblasts and HIMEC, LIPUS significantly decreased the levels of IL-8. The maximum release of EVs, range in size from 100 to 120 nm, was recorded after 60 min in resident cells from both UC and healthy-derived cells. Conversely, PBMC displayed a progressive decrease. In both acute and chronic models, the physiological cellular turnover of the mucosa resulted unchanged after LIPUS. A significative increase of EVs was observed after 2 h of stimulation, and dropped down after 24 h. The levels of EVs were higher (p Conclusion LIPUS proved to be a safe and non pro-inflammatory approach to boost the release of EVs accumulated into the mucosa increasing their levels in the bloodstream. This feature is transient and more evident in the short term. Overall, the LIPUS could pave the way to a new era of a gut disruptive liquid biopsy.

Background Lémann Index (LI) is a well-established validated index to assess cumulative bowel damage in Crohn’s disease (CD). Magnetic resonance imaging and colonoscopy are usually used to measure LI. We assessed the accuracy of bowel ultrasound in assessing LI and its sensitivity to change in a longitudinal cohort of CD patients, in comparison with magnetic resonance imaging. Methods We performed a prospective observational study of 60 patients with active CD. All patients underwent bowel ultrasound, magnetic resonance imaging, and colonoscopy at baseline and at reassessment, within 1 year from treatment with biologics (30 patients were treated with adalimumab, 22 with ustekinumab, four patients with infliximab, and four with vedolizumab). The primary analysis was to determine the correlation between magnetic resonance-based LI and ultrasonography-based LI, both at baseline and at reassessment. Additional analyses established the magnitude of change in LI over time, measured by magnetic resonance imaging and colonoscopy, and its correlation with those in LI measured by bowel ultrasound and colonoscopy. Results The mean values of magnetic resonance-based LI and ultrasonography-based LI at baseline were 6.3 (± 4.8) and 6.4 (± 4.9) respectively, p= 0.39; at reassessment, 5.3 (± 4.6) and 5.5 (± 4.9) respectively, p= 0.30. No differences were observed according to different treatments (Table1). There were significant differences between LI assessed at baseline and LI at reassessment, independently by the imaging tool used for measuring it (p < 0.05). Very high correlations were observed between the magnetic resonance-based LI and ultrasonography-based LI (at baseline, r= 0.964, p < 0.0001; at reassessment, r= 0.970, p < 0.0001) (Figure 1). The magnitude of change in magnetic resonance-based LI correlated with those in ultrasonography-based LI (r= 0.871; p < 0.0001) (Figure 1). Conclusion LI can be measured by bowel ultrasound alternatively to magnetic resonance imaging, without no meaningful change in its value.

Background We assessed the accuracy and the responsiveness to change of Milan Ultrasound Criteria (MUC) in patients with ulcerative colitis (UC), using colonoscopy as the reference standard. Methods We performed a prospective observational study of 49 patients with active UC. All patients underwent bowel ultrasound and colonoscopy at baseline and at reassessment, at week 48, after treatment with biologics (29 patients were treated with infliximab, 14 with vedolizumab, four with adalimumab, two with ustekinumab). Furthermore, patients underwent bowel US also at week 12 and 24. Disease activity was quantified using Mayo endoscopic subscore and MUC. The primary analysis was to determine the accuracy of MUC in identification of endoscopic remission defined as a Mayo endoscopic subscore < 2. Additional analyses established the accuracy of MUC in determining change in lesion severity. Logistic regression was used to examine the relationship between the absence or presence (0/1) of endoscopic remission at reassessment as the dependent variable and possible predictors at week 12 as the independent variables. The model was performed using the stepwise backward method. Results Eighteen patients (37%) got endoscopic remission at reassessment. In Table 1 are summarized endoscopic and ultrasound outcomes achieved at reassessment according to the different treatments. MUC < 6.2 determined endoscopic remission with 83% sensitivity, 84% specificity, 84% accuracy, 75% PVP and 90% PVN (Table 2). MUC was sensitive to change in ulcerative colitis [Guyatt’s responsiveness index: 1.73; standardised effect size ratio: 2.0]. MUC changed significantly from baseline to reassessment [8.72 (7.78−9.14) vs 7.60 (4.71−8.53), p < 0.0001], with the largest drop at week 12 [8.72 (7.82−9.19) vs 7.46 (5.80−7.98), p < 0.0001] (Figure 1). A change of -2 in MUC overtime predicted endoscopic remission at reassessment (AUC 0.806, 95% CI 0.667–0.904; sensitivity 89%, specificity 77%). At multivariable analysis only MUC < 6.2 at week 12 was statistically significantly associated with endoscopic remission at reassessment (Table 3). Conclusion MUC accurately detects endoscopic remission. MUC is a highly responsive, reliable tool for assessing treatment response in patients with ulcerative colitis.

INTRODUCTION PMM2-CDG is the most common congenital disorder of glycosylation (CDGs). It is caused by mutations in the gene PMM2, encoding the enzyme phosphomannomutase-2 (PMM2). A defective PMM2 leads to GDP- mannose deficiency and hypoglycosylation of numerous glycoproteins. At the present, PMM2-CDG has no cure. The use of pharmacological chaperones (PCs) was proposed as a therapeutic approach for many different diseases. PCs are small molecules able to bind unstable target proteins and stabilize them. Since a total lack of phosphomannomutase activity is considered incompatible with life, patients carry at least one misfolding mutation. Thus, PMM2-CDG is a potential candidate for the PC therapy. Phosphomannomutase-2 natural activator in cells is a-glucose-1,6-bisphosphate (aG16P) which stabilizes PMM2 in vitro but it is hydrolysed in vivo by phosphomannomutase-1 (PMM1), particularly in the presence of inosine monophosphate (IMP). OBJECTIVES We are pursuing three different paths in order to stabilize mutant enzymes and to prevent their premature degradation ?Looking for G16P analogs: b-glucose-1,6-bisphosphate (bG16P), the anomer of aG16P and its potential as therapeutic PC ?Improving the cellular uptake of G16P by using lipophilic ?G16P derivative ?Enhancing endogenous ?G16P by knocking out PMM1 METHODS ?-glucose-1-phosphate (?G1P) was synthesized from maltose with bacterial phosphorylase and ?G16P was produced by phosphorylation. ?G16P was purified on an ionic exchange column and was characterized by 31P nuclear magnetic resonance (31P-NMR). In silico docking of ?G16P on PMM2 was carried out by using PELE simulation. Limited proteolysis was performed by incubating PMM2 in the presence of trypsin with or without the ligand and analyzed by SDS-PAGE. Thermal shift assay was used to assess the stabilization in vitro of the recombinant enzyme. The phosphomannomutase activity was monitored by 31P-NMR and by fluorescence spectroscopy. The lipophilic ligand was synthesized by adding acetoxymethyl groups to G16P via standard organic chemistry procedures (collaboration with dott Sodano Univ. of Tourin and prof Rimoli Univ. of Naples). The acetoxymethyl groups would be hydrolyzed once the prodrug is inside the cells. PMM1-KO was edited by CRISPR/Cas9 system in PMM2-CDG fibroblasts.

The era of COVID-19 has brought about a number of novel challenges for the global biobanking community. To better position the biobanking community to cope with current and future challenges, the International Society for Biological and Environmental Repositories (ISBER) COVID-19 Response Task Force was convened to identify needs and gaps in biobanking tools (existing resources that support good practice), for example, standards, best practices, business, etc. and to make recommendations to benefit the community. Toward these goals, the Task Force assembled a set of questions to explore individual biobanks' experiences, with emphasis on identification of key challenges and approaches, including tools employed. A survey was designed with the use of these questions and administered by ISBER. This article presents a summary of the aggregated data obtained from the survey responses, illustrating some of the major issues encountered and identifying which tools the survey respondents found most useful. In particular, this article focuses on the challenges identified during the early months of the COVID-19 era. Recommendations are provided to support biobank emergency preparedness for the future, address lessons learned, and propose solutions to bridge identified gaps. The analysis and the complete survey dataset will also inform the larger Task Force goal to develop specific tool recommendations., info:eu-repo/semantics/publishedVersion

The COVID-19 era has brought about a number of novel challenges for the global biobanking community. An array of diverse tools (e.g., standards, best practices, and plans) exists to support quality and fitness-for-purpose in biobank operations. The International Society for Biological and Environmental Repositories (ISBER) COVID-19 Response Task Force has set out to identify needs and gaps in these tools and make recommendations for the next generation of available tools, having closely examined the COVID-19-related challenges. While conducting this work to examine the relationships between tools and biobank adaptability, a subgroup of the task force conducted a parallel effort to develop and describe individual COVID-19 era case studies based on a number of operating biobanks. Each case study presents a different combination of implemented tools. Observations and lessons learned from these case studies are provided, and experiences with tool implementation are discussed. This information is supplemented by data relating to tool usefulness that was obtained through an ISBER survey discussed in a companion article. The knowledge gained from this study will be combined with other task force efforts to make recommendations to better position the biobanking community in their response to future emergencies., This study was performed by a Task Force organized by members of the International Society for Biological and Environmental Repositories (ISBER) Standards Advisory Committee. The authors gratefully acknowledge our fellow ISBER COVID-19 tools Task Force members who reviewed this article, developed figures and tables, and provided valuable feedback, particularly Daniel Simeon-Dubach, Rebecca Pugh, Sergey Anisimov, and Yehudit Cohen. Shannon McCall and the Duke University BioRepository & Precision Pathology Center receive funding from the United States National Institutes of Health, National Cancer Institute under UM1CA239755 (The Cooperative Human Tissue Network) and P30CA014236 (Duke University’s Cancer Center Support Grant)., info:eu-repo/semantics/publishedVersion

The development of standards for the field of regenerative medicine has been noted as a high priority by several road-mapping activities. Additionally, the U.S. Congress recognizes the importance of standards in the 21st Century Cure Act. Standards will help to accelerate and streamline cell and gene therapy product development, ensure the quality and consistency of processes and products, and facilitate their regulatory approval. Although there is general agreement for the need of additional standards for regenerative medicine products, a shared understanding of standards is required for real progress toward the development of standards to advance regenerative medicine. Here, we describe the roles of standards in regenerative medicine as well as the process for standards development and the interactions of different entities in the standards development process. Highlighted are recent coordinated efforts between the U.S. Food and Drug Administration and the National Institute of Standards and Technology to facilitate standards development and foster science that underpins standards development.

Recent advances in biotechnology are making it possible to advance science and improve healthcare with increasing speed and precision. Biobanking, as a foundation of the biotechnology infrastructure, is critical to the assurance of quality for many of the key components for these advancing technologies in both the human and nonhuman domains. Biobanking must advance to support the increased complexity and required precision needs of biological resources. Standards development can provide an important link for the research and development community by providing tools to ensure quality, fitness-for-purpose, and reproducibility in biobanking. ISBER has been developing the ISBER Best Practices revision. At the same time, ISO/TC276/ WG2 has been developing an International Standard (IS) ISO/DIS 20387 General requirements for biobanking standard. It is important that ISBER and ISO/TC276/WG2 harmonize and/or align their products to enable members of the diverse biobanking community to tailor their own suite of tools to support their specific needs. The availability of both standards and best practices that are complementary will maximize available support for all biobanks. The increased availability of complementary standards, tools, and best practices will facilitate the path to new biotechnology advances and a better future.

A sigma-hole bond is a non-covalent interaction between a covalently-bonded atom of Groups III-VII (donor), bearing a region with a positive electrostatic potential (EP), and a negative site (acceptor). Nowadays, sigma-hole interactions have been found to occur in several organic and biological contexts [1]. The most common and well known sigma-hole bonds are halogen and chalcogen bonds, involving halogen and chalcogen atoms as electrophilic sites, respectively. In the last years, our groups have systematically investigated sigma-hole-dependent enantioseparations, showing that these interactions can actually work in HPLC environment, the occurrence of sigma-bonds being unreported therein [2a-d]. Moreover, envisaging for HPLC on chiral stationary phase (CSP) a novel function other than resolution of racemic mixture, we showed that HPLC and the CSP could be used as technical and molecular tools, respectively, for detection of stereoselective sigma-hole bonds and donors [2e]. On this basis, moving from HPLC towards biological contexts, iodinated 4,4'-bipyridines are currently under investigation as transthyretin (TTR) misfolding inhibitors. In this regard, halogen bonds are reported to underlie ligand-TTR binding, in some cases preventing protein misfolding which is involved in TTR amyloidosis diseases. In our studies, we used in parallel different computational approaches to gather complementary information on structures and recognition mechanisms by correlating experimental and theoretical data, particularly electrostatic potential surface analysis [3a], and molecular dynamics [2c-d,3b]. References [1] (a) Lange, A.; Gu?nther, M.; Bu?ttner, F. M.; Zimmermann, M. O.; Heidrich, J.; Hennig, S.; Zahn, S.; Schall, C.; Sievers-Engler, A.; Ansideri, F.; Koch, P.; Laemmerhofer, M.; Stehle, T.; Laufer, S. A.; Boeckler, F. M. J. Am. Chem. Soc. 2015, 137, 14640-14652; (b) Gilday, L. C. et al. Chem. Rev. 2015, 115, 7118-7195. [2] (a) Peluso, P.; Mamane, V.; Aubert, E.; Cossu, S. J. Chromatogr. A 2014, 1345, 182-192; (b) Peluso, P.; Mamane, V.; Aubert, E.; Dessì, A.; Dallocchio, R. et al. J. Chromatogr. A 2016, 1467, 228-238; (c) Dallocchio, R.; Dessì, A.; Solinas, M.; Arras, A.; Cossu, S.; Aubert, E.; Mamane, V.; Peluso, P. J. Chromatogr. A 2018, 1563, 71-81; (d) Peluso, P.; Gatti, C.; Dessì, A.; Dallocchio, R.; Weiss, R.; Aubert, E.; Pale, P.; Cossu, S.; Mamane, V. J. Chromatogr. A 2018, 1567, 119-129; (e) Peluso, P.; Mamane, V.; Dallocchio, R.; Dessì, A. et al. J. Sep. Sci. 2018, 41, 1247-1256. [3](a) Peluso, P.; Cossu, S. Chirality 2013, 25, 709-718; (b) Peluso, P.; Dessì, A.; Dallocchio, R.; Mamane, V.; Cossu, S. Electrophoresis 2019, DOI: 10.1002/elps.201800493

This paper reviews the major Geomatics solutions available on the market and explores their potentialities for rapid disaster assessment applications. The attention is primarily focused on the most recent satellite and airborne / UAV optical imaging systems, with the goal of providing an update that will assist the selection process when involved in Rapid Mapping. Furthermore, two relevant and on-going experiences, carried out by the authors within international cooperation frameworks, are described with special focus on hazardous events or situations in the Alpine region: (i) Rapid Mapping of mudflow event, by exploiting LiDAR data and satellite, airborne, helicopter- and UAV-based imagery; (ii) multi-temporal monitoring for displacement assessment of landslide event, by integrating multi-sensor and multi-platform Geomatics techniques. Results and lessons learnt are presented that may support the choice of the most suited Geomatics technique.

Vectors derived from adeno-associated virus (AAV) are promising for human gene therapy, including treatment for retinal blindness. One major limitation of AAVs as vectors is that AAV cargo capacity has been considered to be restricted to 4.7 kb. Here we demonstrate that vectors with an AAV5 capsid (i.e., rAAV2/5) incorporated up to 8.9 kb of genome more efficiently than 6 other serotypes tested, independent of the efficiency of the rAAV2/5 production process. Efficient packaging of the large murine Abca4 and human MYO7A and CEP290 genes, which are mutated in common blinding diseases, was obtained, suggesting that this packaging efficiency is independent of the specific sequence packaged. Expression of proteins of the appropriate size and function was observed following transduction with rAAV2/5 carrying large genes. Intraocular administration of rAAV2/5 encoding ABCA4 resulted in protein localization to rod outer segments and significant and stable morphological and functional improvement of the retina in Abca4(-/-) mice. This use of rAAV2/5 may be a promising therapeutic strategy for recessive Stargardt disease, the most common form of inherited macular degeneration. The possibility of packaging large genes in AAV greatly expands the therapeutic potential of this vector system.

Metabolic bone disease associated with primary biliary cirrhosis (PBC) is inadequately characterized. Renal tubular acidosis (RTA) may lead to bone loss through chronic mobilization of skeletal calcium salts to buffer increased acid load.To evaluate the prevalence of RTA in PBC and establish the relationships among bone mineral density (BMD), renal function and nutritional status.We enrolled 69 female patients with compensated PBC and 35 control patients with chronic hepatitis C. RTA was searched in all patients, and 24-h dietary recalls were collected at enrolment. BMD was measured by dual-energy X-ray absorptiometry at the femur neck, lumbar spine and radius ultradistalis sites.No patients received a diagnosis of RTA. BMD values (Z-scores) showed only little deviation from normal population with no difference between PBC and controls. Osteopoenic PBC patients (T-score1) showed significantly lower daily phosphorus intake [median: 672 (288-1374) vs. 921 (253-1923) mg/day; P = 0.037], with a trend towards lower caloric intake than their nonosteopoenic counterparts.Renal tubular acidosis is uncommon in compensated PBC. Cholestasis is not associated with an increased risk of bone demineralization. Inadequate dietary intake may be a preventable factor contributing to bone loss in PBC.

Transient lower oesophageal sphincter relaxation and low lower oesophageal sphincter pressure are the main mechanisms of reflux. It has recently been shown that the stimulation of gamma-aminobutyric acid type B (GABAB) receptors by baclofen decreases the rate of transient lower oesophageal sphincter relaxation and increases the lower oesophageal sphincter pressure in healthy humans. Valproic acid increases synaptosomal GABA concentrations, thus affecting all types of GABA receptors.To evaluate the effect of valproic acid on transient lower oesophageal sphincter relaxation, lower oesophageal sphincter pressure and gastro-oesophageal reflux.Thirteen healthy subjects underwent 2-h post-prandial oesophageal motility and pH monitoring on two separate occasions after the oral administration of 1 g valproic acid or placebo.Valproic acid increased the lower oesophageal sphincter pressure by 41% (14.0 +/- 2.1 mmHg vs. 9.9 +/- 2.0 mmHg after placebo, P0.02), but did not affect the rate of transient lower oesophageal sphincter relaxation (7.9 +/- 1.0/h vs. 8.2 +/- 0.9/h after placebo), the number of reflux episodes or gastro-oesophageal reflux.Non-selective GABA receptor stimulation may be beneficial to reflux patients with low lower oesophageal sphincter pressure, but exerts a different modulation of transient lower oesophageal sphincter relaxation than the selective stimulation of GABAB receptors.

Experiments with two laser radar systems were conducted off the coast of Key West Florida in May of 2001. The purpose of the test was to observe the effect of the water optical properties on the Lidar return signal decay rate and compare the performance of the two systems. The first lidar system, the Shipborad K-meter Survey System (KSS) was configured to transmit linearly polarized light and to receive backscattered light in both channels. The second system, the Airborne KSS, is designed to conduct global surveys from patrolling P3-C aircraft. For this test the Airborne KSS was specially configured to operate from the deck of a ship and both systems were operated in conjunction with each other. The shipboard KSS was configured with a remotely controlled mechanical iris in both receiver channels to allow the use of different fields of view in each channel. Several oceanographic in-situ instruments were used to measure such water properties as optical transmission and absorption, backscatter coefficient, diffuse attenuation , temperature, and salinity as functions of depth. This in-situ dat was then compared with the lidar measurements.

Combined medical treatment may provide further benefit to primary biliary cirrhosis (PBC) patients administered ursodeoxycholic acid (UDCA).To evaluate the long-term effects of colchicine and UDCA in symptomatic PBC patients.We extended up to 10 years the double-blind treatment of 44 symptomatic PBC patients originally included in a 3-year multicentre study comparing UDCA and colchicine (U + C) versus UDCA and placebo (U + P). Outcome measures were death or liver transplantation; incidence of clinically relevant events; clinical and quantitative variables retaining prognostic information.Mean follow-up was 7 +/- 3 years. One patient was lost, three withdrew because of jaundice (U + P); two patients stopped colchicine but remained on UDCA. Eleven patients (two for liver-unrelated reasons, U + P) and six patients (U + C) died, three and two patients, respectively, were transplanted (incidence rate difference, five cases per 100 patient-years; 95% CI, -1 to 11). Hepatocellular carcinoma developed in one (U + P) and four (U + C) patients (difference, -2; CI, -5 to 1), portal hypertension complications in nine patients from each group (difference, 1; CI, -5 to 6). Trends of serum bilirubin, Mayo score, antipyrine clearance were similar among treatment groups.In cirrhotic PBC patients, colchicine does not offer additional benefits to UDCA. In this population, UDCA does not obviate disease progression.

Experiments with a blue-green laser radar system were conducted off the coasts of Ireland and Scotland in June, 1999. The purpose of this test was to measure the effect of the water optical properties on the polarization state and decay rate of the lidar return signal. The lidar system, the K-meter Survey System (KSS), was configured to transmit linearly polarized light and to receive cross-polarized light in one channel and both polarization in the other channel. Several oceanographic ground truth instruments were used to measure the water optical properties, including transmission, absorption, backscatter coefficient, diffuse attenuation, temperature and salinity, as a function of depth. The KSS was mounted on the bow of one of the UK survey vessels, the HMS Roebuck, and the oceanographic instruments were deployed with a deck-mounted winch. The results presented in this paper were obtained both inside and outside of the continental shelf. Since these regions were characterized by different water optical properties, the sensitivity of the lidar return signal in terms of decay rate and polarization to different water clarities was determined.© (1999) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

Many single shot, single pixel underwater LIDAR systems employ high performance photo-multiplier tubes (PMTs) in their receivers. While PMTs offer high gain with a low noise factor, the dynamic range of the output signal is limited by signal induced artifacts. These artifacts include decaying signals with long time constants and short duration `ghost reflection' signals. This paper will characterize the signal-induced artifacts for various single pixel PMTs with different photo-cathode materials and dynode materials.

Testing has been conducted on 8 by 8 avalanche photo diode (APD) arrays derived from large area (16 mm) APDs, both produced by Advanced Photonics, Inc. The array structure was produced using a novel reverse etching process. Tests have been conducted measuring cross- talk, bandwidth, rise and fall times, gain, effective pixel size, and noise characteristics. Measurements have been made as functions of wavelength, optical intensity, and bias voltage. Cross-talk between pixels was characterized under both CW and pulsed (3 nsec) conditions. The effective pixel size was measured by scanning a very small laser spot (.25 mm) across the pixel under test while monitoring the output current. The measured pixel size was approximately 1 mm. This matched very well with the expected physical pixel size of 1 mm. The pulse response was measured by injecting a 3 nsec laser pulse into the pixel under test. The measured response shows that the signal decays approximately 3 orders of magnitude in 60 nsec. The rise time of the pixel is on the order of 5 nsec. Cross-talk between pixels was measured by injecting an optical signal into a pixel. The current output of an adjacent pixel was measrued as the optical power input was increased. The cross-talk a CW optical input is on the order of 1000 to 1. The pulsed cross-talk is on the order of 100 to 1. The cross talk ratio remains constant with varying optical input intensities. The pulsed wavelength response of the APD was characterized at 440 nm and 700 nm. The APD exhibited no difference between the two wavelengths.© (1995) COPYRIGHT SPIE--The International Society for Optical Engineering. Downloading of the abstract is permitted for personal use only.

The Faraday filter is studied for use in a lidar receiver system. The transmission of the Faraday filter is measured as a function of temperature and magnetic field for both the 455 and 459 nm lines of Cs. The measurements are made over a sufficiently wide frequency range in order to determine maximum transmission, hold-off, and effective bandwidth. Various types of polarizers are tested to determine their effect on the filter's field of view (FOV). A comparison between the theoretical model and experimental results is presented. An overall system trade-off study is presented for several different receiver systems.

Titanium carbide, TiC, is one of the hardest materials known (9-10 Mohs), possesses remarkable thermal stability (mp 3067/sup 0/C), and is essentially unaffected by acids and aqueous alkali. These properties make TiC a very useful material for such applications as first-wall coatings for fusion reactors. Unfortunately, crystalline TiC is also brittle, and this limits its structural applications at low temperatures. With present industrial technology, only crystalline TiC coatings can be deposited on complex shapes; the commercial process involves chemical vapor deposition from hydrogen, methane, and titanium tetrachloride at 1000/sup 0/C. More recently, plasma-assisted chemical vapor deposition techniques have been applied to the synthesis of TiC coatings, but these methods also require high temperatures, in excess of 1200/sup 0/C. They now report a simple and powerful chemical vapor deposition (CVD) method for preparing thin films of TiC using an organometallic precursor at exceptionally low temperatures (approx. 150/sup 0/C). Tetraneopentyltitanium, Ti(CH/sub 2/C(CH/sub 3/)/sub 3/)/sub 4/ was chosen for metal-organic chemical vapor deposition (MOCVD) studies since it volatilizes easily and has been reported to thermolyze at low temperature. They have devised an improved synthesis of Ti(CH/sub 2/C(CH/sub 3/)/sub 3/)/sub 4/: interaction of Ti(OCH/sub 2/CH/sub 3/)/sub 4/ with 4 equi of LiCH/sub 2/C(CH/sub 3/)/submore » 3/ in pentane followed by sublimation at 50/sup 0/C (10/sup -3/ torr) gives yellow crystals of the titanium alkyl.« less

Titanium carbide, TiC, is one of the hardest materials known (9-10 Mohs), possesses remarkable thermal stability (mp 3067/sup 0/C), and is essentially unaffected by acids and aqueous alkali. These properties make TiC a very useful material for such applications as first-wall coatings for fusion reactors. Unfortunately, crystalline TiC is also brittle, and this limits its structural applications at low temperatures. With present industrial technology, only crystalline TiC coatings can be deposited on complex shapes; the commercial process involves chemical vapor deposition from hydrogen, methane, and titanium tetrachloride at 1000/sup 0/C. More recently, plasma-assisted chemical vapor deposition techniques have been applied to the synthesis of TiC coatings, but these methods also require high temperatures, in excess of 1200/sup 0/C. They now report a simple and powerful chemical vapor deposition (CVD) method for preparing thin films of TiC using an organometallic precursor at exceptionally low temperatures (approx. 150/sup 0/C). Tetraneopentyltitanium, Ti(CH/sub 2/C(CH/sub 3/)/sub 3/)/sub 4/ was chosen for metal-organic chemical vapor deposition (MOCVD) studies since it volatilizes easily and has been reported to thermolyze at low temperature. They have devised an improved synthesis of Ti(CH/sub 2/C(CH/sub 3/)/sub 3/)/sub 4/: interaction of Ti(OCH/sub 2/CH/sub 3/)/sub 4/ with 4 equi of LiCH/sub 2/C(CH/sub 3/)/submore » 3/ in pentane followed by sublimation at 50/sup 0/C (10/sup -3/ torr) gives yellow crystals of the titanium alkyl.« less

Background: Gene transfer using adeno-associated viral (AAV) vectors has been successfully applied in the retina for the treatment of inherited retinal dystrophies. Recently, microRNAs have been exploited to fine-tune transgene expression improving therapeutic outcomes. Here we evaluated the ability of retinal-expressed microRNAs to restrict AAV-mediated transgene expression to specific retinal cell types that represent the main targets of common inherited blinding conditions. Methodology/Principal Findings: To this end, we generated AAV2/5 vectors expressing EGFP and containing four tandem copies of miR-124 or miR-204 complementary sequences in the 3′UTR of the transgene expression cassette. These vectors were administered subretinally to adult C57BL/6 mice and Large White pigs. Our results demonstrate that miR-124 and miR-204 target sequences can efficiently restrict AAV2/5-mediated transgene expression to retinal pigment epithelium and photoreceptors, respectively, in mice and pigs. Interestingly, transgene restriction was observed at low vector doses relevant to therapy. Conclusions: We conclude that microRNA-mediated regulation of transgene expression can be applied in the retina to either restrict to a specific cell type the robust expression obtained using ubiquitous promoters or to provide an additional layer of gene expression regulation when using cell-specific promoters. © 2011 Karali et al.

Vectors based on the adeno-associated virus (rAAV) are able to transduce the retina of animal models, including non-human primates, for a long-term period, safely and at sustained levels. The ability of the various rAAV serotypes to transduce retinal target cells has been exploited to successfully transfer genes to photoreceptors, retinal pigment epithelium and the inner retina, which are affected in many inherited and non-inherited blinding diseases. rAAV-mediated, constitutive and regulated gene expression at therapeutic levels has been achieved in the retina of animal models, thus providing proof-of-principle of gene therapy efficacy and safety in models of dominant and recessive retinal disorders. In addition, gene transfer of molecules with either neurotrophic or antiangiogenic properties provides useful alternatives to the classic gene replacement for treatment of both mendelian and complex traits affecting the retina. Years of successful rAAV-mediated gene transfer to the retina have resulted in restoration of vision in dogs affected with congenital blindness. This has paved the way to the first attempts at treating inherited retinal diseases in humans with rAAV. Although the results of rAAV clinical trials for non-retinal diseases give a warning that the outcome of viral-mediated gene transfer in humans may be different from that predicted based on results in other species, the immune privilege of the retina combined with the versatility of rAAV serotypes may ultimately provide the first successful treatment of human inherited diseases using rAAV.

Autosomal dominant retinitis pigmentosa caused by the frequent rhodopsin P23H mutation is characterized by progressive photoreceptor cell death eventually leading to blindness and for which no therapies are available. Considering the gain-of-function effect exerted by the P23H mutation, strategies aimed at silencing the expression of the mutated allele, like RNA interference, are desirable. We have designed small interfering RNAs (siRNA) to silence specifically the P23H rhodopsin allele expressed by a transgenic rat model of the disease. We have selected in vitro one siRNA and generated an adeno-associated viral (AAV) vector expressing the short hairpin RNA (shRNA) based on the selected siRNA. In vitro the shRNA significantly inhibits the expression of the P23H but not the wild-type rhodopsin allele. Subretinal administration of the AAV2/5 vector encoding the shRNA in P23H transgenic rats results in inhibition of rhodopsin P23H expression that is not able to prevent or block photoreceptor degeneration. Since rhodopsin is the most abundant rod photoreceptor protein, systems resulting in more robust shRNA expression in the retina may be required to achieve therapeutic efficacy in vivo.

Molecules with neurotrophic activity are being evaluated for treatment of retinitis pigmentosa in animal models. In particular, great interest has been focused recently on erythropoietin (Epo). Evidence of its neurotrophic activity comes mainly from data demonstrating photoreceptor protection in a rodent light-damage model through systemic administration of a recombinant form of this hormone. Our goal was to test whether Epo retinal gene transfer can rescue or delay photoreceptor cell death. We delivered adeno-associated viral vectors encoding Epo intraocularly and, for comparison, intramuscularly to one light-induced and two genetic models of retinal degeneration. Intraocular Epo gene transfer resulted in sustained hormone expression in the eye, which was undetectable systemically. In contrast, Epo intramuscular gene transfer resulted in hormone secretion in the circulation, which was not detected in ocular fluids. The protein secreted from muscle and retina is of the same molecular weight as a commercial recombinant human Epo. Interestingly, following systemic but not intraocular Epo delivery, morphological photoreceptor protection was observed in the light-damage and rds/peripherin (Prph2) models of retinal degeneration. In the light-damage model, the morphological rescue was accompanied by a significant electrophysiological improvement of photoreceptor function. In contrast, no photoreceptor rescue was observed following Epo gene transfer in the rd10 model. This suggests that different apoptotic mechanisms, with varying sensitivities to Epo, occur in different retinal degeneration models. In conclusion, our data support Epo as a neuroprotective agent in some, but not all, retinal degenerations. Further, rescue is observed in specific models after systemic but not intraocular Epo gene transfer.

Purpose Mutations in the PDE6B gene cause recessive, severe retinitis pigmentosa (RP). PDE6B encodes the β subunit of the rod-specific phosphodiesterase (βPDE), which, when absent, results in toxic levels of intracellular Ca(2+) and photoreceptor cell death. Ca(2+) blockers, such as nilvadipine, as well as light restriction, slow photoreceptor degeneration in animal models of βPDE deficiencies. The goal of the study was to evaluate the efficacy of AAV2/5- or AAV2/8-mediated gene replacement in combination with nilvadipine and/or with light restriction in the rd10 mouse bearing homozygous pde6b mutations. Methods AAV vectors encoding either βPDE or EGFP were subretinally administered at postnatal day (P)2. Nilvadipine was administered from P7 to P28. For light restriction, pregnant rd10 mice were kept in a dark environment until their pups were 28 days old. All functional and histologic analyses were performed at P35. Results Significant morphologic photoreceptor protection was observed after subretinal administration of AAV vectors encoding EGFP. This protection further increased after administration of AAV2/8 or -2/5 encoding for βPDE and was not associated with significant functional improvement. Photoreceptor protection was higher after AAV2/8- than after AAV2/5-mediated delivery and was not significantly augmented by additional drug therapy and/or light restriction. The protective effect was lost after P35. Conclusions In conclusion, more efficient gene transfer tools than those used in this study, as well as a better understanding of the disease pathogenesis, should be explored to increase the effect of gene replacement and to design gene-based strategies that block the apoptotic pathways activated by βPDE deficiency.