Reduction of choroidal neovascularization in mice by adeno-associated virus-delivered anti-vascular endothelial growth factor short hairpin RNA.

BACKGROUND: Strategies leading to the long-term suppression of inappropriate ocular angiogenesis are required to avoid the need for repetitive monthly injections for treatment of diseases of the eye, such as age-related macular degeneration (AMD). The present study aimed to develop a strategy for the sustained repression of vascular endothelial growth factor (VEGF), which is identified as the key player in exudative AMD. METHODS: We have employed short hairpin (sh)RNAs combined with adeno-associated virus (AAV) delivery to obtain the targeted expression of potent gene-regulatory molecules. Anti-VEGF shRNAs were analyzed in human retinal pigment epithelial (RPE) cells using Renilla luciferase screening. For in vivo delivery of the most potent shRNA, self-complementary AAV vectors were packaged in serotype 8 capsids (scAAV2/8-hU6-sh9). In vivo efficacy was evaluated either by injection of scAAV2/8-hU6-sh9 into murine hind limb muscles or in a laser-induced murine model of choroidal neovascularization (CNV) following scAAV2/8-hU6-sh9 subretinal delivery. RESULTS: Plasmids encoding anti-VEGF shRNAs showed efficient knockdown of human VEGF in RPEs. Intramuscular administration led to localized expression and 91% knockdown of endogenous murine (m)VEGF. Subsequently, the ability of AAV2/8-encoded shRNAs to impair vessel formation was evaluated in the murine model of CNV. In this model, the sizes of the CNV were significantly reduced (up to 48%) following scAAV2/8-hU6-sh9 subretinal delivery. CONCLUSIONS: Using anti-VEGF vectors, we have demonstrated efficient silencing of endogenous mVEGF and showed that subretinal administration of scAAV2/8-hU6-sh9 has the ability to impair vessel formation in an AMD animal model. Thus, AAV-encoded shRNA can be used for the inhibition of neovascularization, leading to the development of sustained anti-VEGF therapy. Copyright © 2012 John Wiley & Sons, Ltd.

Short and longterm ocular vascular effects after intravitreal injection of ranibizumab for neovascular age-related macular degeneration

Purpose: To investigate the effect of the first and repeated intravitreal injections of ranibizumab (1.25mg; 0.05ml) on retrobulbar blood flow velocities in patients with wet age-related macular degeneration (AMD). Methods: This prospective non randomized study included twenty consecutive AMD patients. Time- averaged mean blood flow velocities (BFVs) in the central retinal, temporal posterior ciliary and ophthalmic arteries (CRA, TPCA and OA) were measured by ultrasound imaging before, 2 days and 3 weeks after the first injection of ranibizumab, then 6 months after supplemental monthly injections if required. At each visit, complete ophthalmological examination was performed, including best corrected visual acuity measurement according to ETDRS protocol and OCT. Results: In the treated eyes, ranibizumab injection was followed by a significant improvement in visual acuity (from 44.4 ± 21.7, to 50.9±25.9 (p<0.01) at month 6, and a decrease in mean central macular thickness from 377±115 to 267 ± 74 µm (p<0.001) at month 6. At day 2 mean BFVs decreased by 16% in the CRA and by 20% in TPCA (p<0.001, both), then remained stable. Mean BFVs did not change in OA at the day 2 but decreased at week 3 by 18% (p<0.001). Supplemental injections did not lead to additional effects at month 6. No effect was tabulated in the fellow eye. Conclusions: We report an early decrease in mean BFV in CRA and TPRA following intravitreal injections of ranibizumab corresponding to vasoconstrictive effect of this drug. Decrease in mean BFV in all retrobulbar arteries from the week 3 suggests that ranibizumab proceeds to a local and regional vasoconstrictive and antiangiogenic effects after local diffusion. Thus, ranibizumab could induce an actual hypoperfusion of the treated eye which could correspond to a vascular side effect.

Effects of a short-term overfeeding with fructose or glucose in healthy young males.

Consumption of simple carbohydrates has markedly increased over the past decades, and may be involved in the increased prevalence in metabolic diseases. Whether an increased intake of fructose is specifically related to a dysregulation of glucose and lipid metabolism remains controversial. We therefore compared the effects of hypercaloric diets enriched with fructose (HFrD) or glucose (HGlcD) in healthy men. Eleven subjects were studied in a randomised order after 7 d of the following diets: (1) weight maintenance, control diet; (2) HFrD (3.5 g fructose/kg fat-free mass (ffm) per d, +35 % energy intake); (3) HGlcD (3.5 g glucose/kg ffm per d, +35 % energy intake). Fasting hepatic glucose output (HGO) was measured with 6,6-2H2-glucose. Intrahepatocellular lipids (IHCL) and intramyocellular lipids (IMCL) were measured by 1H magnetic resonance spectroscopy. Both fructose and glucose increased fasting VLDL-TAG (HFrD: +59 %, P < 0.05; HGlcD: +31 %, P = 0.11) and IHCL (HFrD: +52 %, P < 0.05; HGlcD: +58 %, P = 0.06). HGO increased after both diets (HFrD: +5 %, P < 0.05; HGlcD: +5 %, P = 0.05). No change was observed in fasting glycaemia, insulin and alanine aminotransferase concentrations. IMCL increased significantly only after the HGlcD (HFrD: +24 %, NS; HGlcD: +59 %, P < 0.05). IHCL and VLDL-TAG were not different between hypercaloric HFrD and HGlcD, but were increased compared to values observed with a weight maintenance diet. However, glucose led to a higher increase in IMCL than fructose.

Harmonisation of short-term in vitro culture for the expansion of antigen-specific CD8(+) T cells with detection by ELISPOT and HLA-multimer staining.

Ex vivo ELISPOT and multimer staining are well-established tests for the assessment of antigen-specific T cells. Many laboratories are now using a period of in vitro stimulation (IVS) to enhance detection. Here, we report the findings of a multi-centre panel organised by the Association for Cancer Immunotherapy Immunoguiding Program to investigate the impact of IVS protocols on the detection of antigen-specific T cells of varying ex vivo frequency. Five centres performed ELISPOT and multimer staining on centrally prepared PBMCs from 3 donors, both ex vivo and following IVS. A harmonised IVS protocol was designed based on the best-performing protocol(s), which was then evaluated in a second phase on 2 donors by 6 centres. All centres were able to reliably detect antigen-specific T cells of high/intermediate frequency both ex vivo (Phase I) and post-IVS (Phase I and II). The highest frequencies of antigen-specific T cells ex vivo were mirrored in the frequencies following IVS and in the detection rates. However, antigen-specific T cells of a low/undetectable frequency ex vivo were not reproducibly detected post-IVS. Harmonisation of the IVS protocol reduced the inter-laboratory variation observed for ELISPOT and multimer analyses by approximately 20 %. We further demonstrate that results from ELISPOT and multimer staining correlated after (P < 0.0001 and R (2) = 0.5113), but not before IVS. In summary, IVS was shown to be a reproducible method that benefitted from method harmonisation.

Indexing strategies for rapid searches of short words in genome sequences.

Searching for matches between large collections of short (14-30 nucleotides) words and sequence databases comprising full genomes or transcriptomes is a common task in biological sequence analysis. We investigated the performance of simple indexing strategies for handling such tasks and developed two programs, fetchGWI and tagger, that index either the database or the query set. Either strategy outperforms megablast for searches with more than 10,000 probes. FetchGWI is shown to be a versatile tool for rapidly searching multiple genomes, whose performance is limited in most cases by the speed of access to the filesystem. We have made publicly available a Web interface for searching the human, mouse, and several other genomes and transcriptomes with oligonucleotide queries.

Unplanned nursing home short stay : elderly patients characteristics and 3-month outcomes.

Background: Nursing home short stays (NHSS) in the canton of Vaud have been introduced for respite care purpose. However, a growing number of older patients are urgently admitted from home (within 24h) or directly after hospital discharge (58% of all admissions in 2010). NHSS appears therefore as an increasingly important component of the health care system, but the characteristics of admitted patients have not been previously described. A better knowledge would contribute to identify specific care needs and enhance their care. Objectives: 1) To describe the characteristics of patients admitted in unplanned NHSS ( after hospital stay or urgently from home); 2) To determine living disposition 3-month after NHSS discharge. Method: Over a 18-month period, elderly patients with unplanned NHSS admission to 2 facilities in Lausanne were identified. Demographic, social, health, and functional data, as well as main reason for admission were collected. Death and place of living at 3-months were collected using the administrative database. Results: Overall, 114 patients (mean age 83.1 ± 6.2 years, 77% women, 84% living alone) were assessed, 80% being admitted from hospital. Mean score in Lawton's instrumental ADL before NHSS admission was 4.6 ± 2.5 and 69% of the patients were home care recipients (median number of weekly visits: 5 ± 3). Patients reported going out 4.2 ± 1.3 times/week and 56% reported at least one fall over the past year. Among the 91 patients coming from the hospital, main reason for admission was injury/limb immobilization (58%), recuperation (13%) and functional impairment in basic ADL (10%). Mean score at Katz's Basic ADL at admission was 3.7 ± 1.9. Overall, 90% of patients were identified with gait and balance impairment, 78% with cognitive impairment and 70% with polypharmacy (>6 different drugs). At 3-month after NHSS discharge (N = 92), 72% patients were living at home, 16% had been admitted to long term care, and 6% died. Among patients living at home at follow-up, 11% had been readmitted to hospital during the follow-up period. Conclusion: Older patients with unplanned NHSS admission show a high prevalence of functional, mobility, and cognitive impairments, as well as other geriatric syndromes. Specific measures should be considered during these stays to prevent further functional decline and, possibly, hospital readmission. Patients admitted with basic ADLs impairment might be candidate for higher levels of care (rehabilitation).

Assimilation des expériences problématiques: une étude de cas de psychothérapie psychodynamique limitée dans le temps [Assimilation of problematic experiences: a case study on short-term dynamic psychotherapy].

The assimilation model is a qualitative and integrative approach that enables to study change processes that occur in psychotherapy. According to Stiles, this model conceives the individual's personality as constituent of different voices; the concept of voice is used to describe traces left by past experiences. During the psychotherapy, we can observe the progressive integration of the problematic voices into the patient's personality. We applied the assimilation model to a 34-session-long case of an effective short-term dynamic psychotherapy. We've chosen eight sessions we transcribed and analyzed by establishing points of contact between the case and the theory. The results are presented and discussed in terms of the evolution of the main voices in the patient.

Short-term stability of testosterone and epitestosterone conjugates in urine samples: quantification by liquid chromatography-linear ion trap mass spectrometry.

A simple method using liquid chromatography-linear ion trap mass spectrometry for simultaneous determination of testosterone glucuronide (TG), testosterone sulfate (TS), epitestosterone glucuronide (EG) and epitestosterone sulfate (ES) in urine samples was developed. For validation purposes, a urine containing no detectable amount of TG, TS and EG was selected and fortified with steroid conjugate standards. Quantification was performed using deuterated testosterone conjugates to correct for ion suppression/enhancement during ESI. Assay validation was performed in terms of lower limit of detection (1-3ng/mL), recovery (89-101%), intraday precision (2.0-6.8%), interday precision (3.4-9.6%) and accuracy (101-103%). Application of the method to short-term stability testing of urine samples at temperature ranging from 4 to 37 degrees C during a time-storage of a week lead to the conclusion that addition of sodium azide (10mg/mL) is required for preservation of the analytes.

Rivascolarizzazione miocardica con arteria radiale: tecniche chirurgiche a confronto e risultati a breve termine [Myocardial revascularization and radial artery: different surgical strategies and short-term results]

BACKGROUND: The radial artery is routinely used as a graft for surgical arterial myocardial revascularization. The proximal radial artery anastomosis site remains unknown. In this study, we analyzed the short-term results and the operative risk determinants after having used four different common techniques for radial artery implantation. METHODS: From January 2000 to December 2004, 571 patients underwent coronary artery bypass grafting with radial arteries. Data were analyzed for the entire population and for subgroups following the proximal radial artery anastomosis site: 140 T-graft with the mammary artery (group A), 316 free-grafts with the proximal anastomosis to the ascending aorta (group B), 55 mammary arteries in situ elongated with the radial artery (group C) and 60 radial arteries elongated with a piece of mammary artery and anastomosed to the ascending aorta (group D). RESULTS: The mean age was 53.8 +/- 7.7 years; 55.5% of patients had a previous myocardial infarction and 73% presented with a satisfactory left ventricular function. A complete arterial myocardial revascularization was achieved in 532 cases (93.2%) and 90.2% of the procedures were performed under cardiopulmonary bypass and cardioplegic arrest. The operative mortality rate was 0.9%, a postoperative myocardial infarction was diagnosed in 19 patients (3.3%), an intra-aortic balloon pump was used in 10 patients (1.7%) and a mechanical circulatory device was implanted in 2 patients. The radial artery harvesting site remained always free from complications. The proximal radial artery anastomosis site was not a determinant of early hospital mortality. Group C showed a higher risk of postoperative myocardial infarction (p = 0.09), together with female gender (p = 0.003), hypertension (p = 0.059) and a longer cardiopulmonary bypass time. CONCLUSIONS: The radial artery and the mammary artery can guarantee multiple arterial revascularization also for patients with contraindications to double mammary artery use. The four most common techniques for proximal radial artery anastomosis are not related to a higher operative risk and they can be used alternatively to reach the best surgical results

Prolonged but not short negative energy condition restored corticoadrenal leptin sensitivity in the hypothalamic obese rat.

BACKGROUND/AIM: We have reported that neonatal treatment with monosodium L-glutamate (MSG), which causes damage to the arcuate nucleus, leads to severe hyperleptinemia and reduced adrenal leptin receptor (ob-Rb) expression in adulthood. As a result, rats given MSG neonatally display corticoadrenal leptin-resistance, a defect that is overridden by normalization of corticoadrenal hyperfunction. The aim of the present study was to determine whether negative energy conditions could correct corticoadrenal cell dysfunction in rats given MSG neonatally. METHODS: Normal (CTR) and MSG-treated female rats were subjected to food removal for 1-5 days, or prolonged (24-61 days) food restriction (FR). Plasma levels of several biomarkers and in vitro corticoadrenal function were evaluated following starvation or FR. RESULTS: Fasting for 1-5 days reduced plasma leptin levels in CTR and MSG rats, compared to levels in the respective groups fed ad libitum(p < 0.05), but adrenal leptin-resistance was unchanged. With prolonged FR, isolated adrenal cells from MSG rats became sensitive to leptin, which lowered ACTH-induced glucocorticoid release. This restoration of leptin response was associated with normalization of adrenal ob-Rb gene expression. CONCLUSION: Dietary restriction in some leptin-resistant obese phenotypes may normalize adrenocortical function.