Efficient gene delivery to the cone-enriched pig retina by dual AAV vectors.

Gene therapy with adeno-associated viral (AAV) vectors is limited by AAV cargo capacity that prevents their application to the inherited retinal diseases (IRDs), such as Stargardt disease (STGD) or Usher syndrome type IB (USH1B), which are due to mutations in genes larger than 5 kb. Trans-splicing or hybrid dual AAV vectors have been successfully exploited to reconstitute large gene expression in the mouse retina. Here, we tested them in the large cone-enriched pig retina that closely mimics the human retina. We found that dual AAV trans-splicing and hybrid vectors transduce pig photoreceptors, the major cell targets for treatment of IRDs, to levels that were about two- to threefold lower than those obtained with a single AAV vector of normal size. This efficiency is significantly higher than that in mice, and is potentially due to the high levels of dual AAV co-transduction we observe in pigs. We also show that subretinal delivery in pigs of dual AAV trans-splicing and hybrid vectors successfully reconstitute, albeit at variable levels, the expression of the large genes ABCA4 and MYO7A mutated in STGD and USH1B, respectively. Our data support the potential of dual AAV vectors for large gene reconstitution in the cone-enriched pig retina that is a relevant preclinical model.

Retinal transduction profiles by high-capacity viral vectors.

Retinal gene therapy with adeno-associated viral (AAV) vectors is safe and effective in humans. However, the limited cargo capacity of AAV prevents their use for therapy of those inherited retinopathies (IRs) due to mutations in large (>5 kb) genes. Viral vectors derived from adenovirus (Ad), lentivirus (LV) and herpes virus (HV) can package large DNA sequences, but do not target efficiently retinal photoreceptors (PRs) where the majority of genes responsible for IRs are expressed. Here, we have evaluated the mouse retinal transduction profiles of vectors derived from 16 different Ad serotypes, 7 LV pseudotypes and from a bovine HV. Most of the vectors tested transduced efficiently the retinal pigment epithelium. We found that LV-GP64 tends to transduce more PRs than the canonical LV-VSVG, albeit this was restricted to a narrow region. We observed more extensive PR transduction with HdAd1, 2 and 5/F35++ than with LV, although none of them outperformed the canonical HdAd5 or matched the extension of PR transduction achieved with AAV2/8.

[The 2013 Seville Consensus Document on alternatives to allogenic blood transfusion. An update on the Seville Document]. / 2013. Documento Sevilla de Consenso sobre Alternativas a la Transfusión de Sangre Alogénica. Actualización del Documento Sevilla.

Since allogeneic blood transfusion (ABT) is not harmless, multiple alternatives to ABT (AABT) have emerged, though there is great variability in their indications and appropriate use. This variability results from the interaction of a number of factors, including the specialty of the physician, knowledge and preferences, the degree of anemia, transfusion policy, and AABT availability. Since AABTs are not harmless and may not meet cost-effectiveness criteria, such variability is unacceptable. The Spanish Societies of Anesthesiology (SEDAR), Hematology and Hemotherapy (SEHH), Hospital Pharmacy (SEFH), Critical Care Medicine (SEMICYUC), Thrombosis and Hemostasis (SETH) and Blood Transfusion (SETS) have developed a Consensus Document for the proper use of AABTs. A panel of experts convened by these 6 Societies have conducted a systematic review of the medical literature and have developed the 2013 Seville Consensus Document on Alternatives to Allogeneic Blood Transfusion, which only considers those AABT aimed at decreasing the transfusion of packed red cells. AABTs are defined as any pharmacological or non-pharmacological measure aimed at decreasing the transfusion of red blood cell concentrates, while preserving patient safety. For each AABT, the main question formulated, positively or negatively, is: « Does this particular AABT reduce the transfusion rate or not?¼ All the recommendations on the use of AABTs were formulated according to the Grades of Recommendation Assessment, Development and Evaluation (GRADE) methodology.

Surgical technique for laparoscopic removal of bulky para-aortic nodes without repositioning surgical field during laparoscopic debulking for advanced ovarian cancer.

Background: In the last years, laparoscopy has been progressively introduced in the management of advanced- stage ovarian cancer (AOC) not only to evaluate tumour resectability, but also to perform primary or interval minimally invasive debulking surgery in selected patients. During laparoscopic debulking for AOC, the need to change the surgical field to treat disease in the upper abdomen can be a time-consuming procedure. Objective: To demonstrate feasibility, safety and effectiveness of laparoscopic approach to remove bulky para- aortic nodes in AOC with a 30-degree 3D-endoscope without repositioning the laparoscopic surgical field. Materials and Methods: A 51-year-old woman was referred to our centre due to AOC with bulky para-aortic nodes (7 cm polylobate mass at CT-scan). The narrated surgical video article demonstrates the surgical steps for laparoscopic removal of bulky para-aortic nodes with a 30-degree 3D-endoscope, maintaining the vision from the upper abdomen perpendicular to the main axis of the vascular structures for the whole duration of the surgery ("top-bottom" view), without repositioning surgical field. Main Outcomes measures: Complete laparoscopic excision of disease was achieved. Results: Post-operative course was uneventful. Patient recovered from surgery and was able to start adjuvant chemotherapy within 30 days from surgery. Conclusions: Repositioning the surgical field to perform para-aortic dissection can be a time-consuming procedure during laparoscopic debulking for ovarian cancer. Laparoscopic removal of bulky para-aortic nodes with a 30-degree 3D-endoscope and "top-bottom view" is feasible, safe and effective.

Diagnostic accuracy and economic impact of three work-up strategies identifying risk groups in endometrial cancer, fully incorporating sentinel lymph node algorithm.

BACKGROUND: According to the European Society for Medical Oncology/ European Society of Gynaecological Oncology/European Society for Radiotherapy and Oncology (ESMO/ESGO/ESTRO) Consensus Conference, the role of preoperative risk groups (RGs) in endometrial cancer (EC) is to direct surgical nodal staging. We compared diagnostic accuracy and economic impact of three work-up strategies to identify RGs. METHODS: A retrospective multicentre study including patients with early-stage EC. The three different work-up strategies were as follows:-Mondovì Hospital: transvaginal ultrasonography, pelvic magnetic resonance imaging (MRI); frozen section examination of the uterus in case of imaging discordance. High-risk patients underwent abdominal computed tomography.-Gemelli Hospital: transvaginal ultrasonography, MRI, One-Step Nucleic Acid Amplification (OSNA) of sentinel lymph node (SLN); frozen section examination of the uterus in case of imaging discordance.-Negrar Hospital: positron emission tomography (PET), frozen section examination of the uterus and of SLN. For statistical purposes patients were assigned, preoperatively and postoperatively, to two groups: group A (high-risk) and group B (not high-risk). RESULTS: Three hundred eighty-five patients were included (93 Mondovì, 215 Gemelli, 77 Negrar). Endometrial biopsy errors led to 47.3% misclassifications. Test accuracy of Mondovì, Gemelli and Negrar strategies was 0.83 (95%CI 0.734-0.901), 0.95 (95%CI 0.909-0.975) and 0.94 (95%CI 0.866-0.985), respectively. Preoperative work-up mean cost per patient in group A was €514.5 at Mondovì, €868.5 at Gemelli, and €1212.8 at Negrar hospital (p-value < 0.001), while in group B was €378.8 at Mondovì, €941.2 at Gemelli, and €1848.4 at Negrar hospital (p-value < 0.001). CONCLUSIONS: In our study, work-up strategies with more relevant economic impact showed a better diagnostic accuracy. Upcoming guidelines should specify recommendations about the gold standard work-up strategy, including the role of SLN.

The gluteal fold fascio-cutaneous flap for reconstruction after radical excision of primary vulvar cancers.

OBJECTIVES: Reconstructive surgery plays an important role in cosmetic and functional results of major excisional surgery performed as a treatment for invasive vulvar cancer. Traditional techniques -- gracilis myocutaneous o rectus abdominis flaps -- have several limits. We describe here a different surgical approach that we have used since 1998 in an effort to obtain better results in vulvar reconstruction. METHODS: From January 1998 to June 2007, thirty three patients who underwent excisional radical surgery for invasive vulvar tumors, were treated with vulvar reconstruction using the gluteal fold fascio-cutaneous local flap. Flaps were designed along the gluteal fold in adequate length and size. They were oval or triangular in shape depending on the defect they were supposed to cover. The flaps -- which always included the fascial layer -- were raised up to identify a perforator branch of the internal pudendal artery and then harvested as an island flap to achieve better mobility. RESULTS: We had no major complications, only two patients presented marginal necrosis and eight patients experienced significant seromas. Advantages over the alternative techniques included reduced dimensions of scars, absence of flap liponecrosis, no need of modifying patient's position on the surgical table, and very limited blood loss. CONCLUSIONS: We conclude that gluteal fold flap offers excellent cosmetic and functional results with a low complication rate. Therefore we support the gluteal fold flap as a valid surgical option whenever reconstruction is needed after radical excision of vulvar neoplasms.

Effect of nitrite upon leghemoglobin and interaction with nitrogen fixation.

Nitrite (0.4 mM) added to soybean bacteroid preparations strongly inhibited C2H2 reduction. In the presence of leghemoglobin (0.1mM), a 3-fold enhancement of nitrogen fixation occurred but the inhibitory effect of nitrite was delayed. Spectra of leghemoglobin showed a rapid disappearance of the 574 nm and 541 nm peaks of oxyleghemoglobin the presence of nitrite. Concomitant oxidation of this hemoprotein gave ferric leghemoglobin as the single final product. High nitrite levels could depress nitrogen fixation both by inactivation of nitrogenase and by conversion of leghemoglobin into an inactive form. Nitrite present at low concentrations reacts with this hemoprotein and is then no longer able to penetrate into bacteroids.

The reactivity of thiol compounds with different redox states of leghaemoglobin: evidence for competing reduction and addition pathways.

Reaction of the ferric form of leghaemoglobin with hydrogen peroxide has been previously shown to give rise to an iron(IV)-oxo (ferryl) species, and a protein radical. Inclusion of a variety of thiol compounds in this system is shown to lead to rapid loss of the iron(IV)-oxo species and the regeneration of the ferric form and/or the formation of novel sulf species formed by nucleophilic attack of the thiol group on the tetrapyrrole ring. The reduction process also results in the generation of thiyl radicals which have been detected by EPR spin trapping. The relative yields of the products produced by these two competing pathways is shown to be highly dependent on the steric and electronic characteristics of the thiol compound. Evidence has also been obtained, in the absence of hydrogen peroxide, for both the reduction of the ferric form of the protein to the oxy-ferrous form, via a process believed to involve the deoxy-ferrous species, and the formation of sulf-leghaemoglobin species. Both of these pathways are again highly dependent on the structure of the thiol, and the former also results in the generation of thiyl radicals. Inclusion of the sulfide anion in place of the organic thiols results in somewhat different behaviour, in that this species appears to both reduce the iron centre and form a complex with the iron atom. This ligation process is reversible, and the sulfide complex is shown to react readily with both strong oxidizing and reducing agents. The behaviour of this protein, which is structurally related to myoglobin, is dramatically different to that demonstrated by myoglobin; this is rationalized in terms of the much more open heme site of leghaemoglobins, and the presence of an electronic gate which hinders access by negatively charged molecules. The contribution of these processes to the maintenance of the leghaemoglobin proteins in the oxy-ferrous form in vivo and the binding of oxygen is discussed.

Identification of the site of the globin-derived radical in leghaemoglobins.

Reaction of the Fe3+ form of the oxygen-carrying protein leghaemoglobin (MetLb), derived from the root nodules of lupins, with H2O2 is shown to generate, in addition to an iron (IV)-oxo (ferryl) species, a globin radical. This radical has been detected by EPR spectroscopy and is analogous to the species previously observed with the soybean protein. Analysis of the hyperfine coupling constants and g value of the EPR signal, together with computer simulations and the similarity of the observed spectra of that detected with the soybean form suggest that this species is also a tyrosine-derived phenoxyl radical; this species is believed to arise via an electron-transfer process within the protein with an electron being transferred from the tyrosine residue to an initially-generated Compound-1-type species. Comparison of the protein sequences and structures of the two proteins show that there is only one conserved tyrosine residue (at position 133 in the soybean and 138 in the lupin); this is believed to be the site of the phenoxyl radical. The lupin phenoxyl radical reacts with added water-soluble antioxidants and reducing agents which result in repair of the radical; this may be an important protective mechanism in vivo. Analysis of molecular models of the protein structures is in accord with both the assignment of the radical to this conserved tyrosine residue and the observed radical reactivity.

Reaction of ferric leghemoglobin with H2O2: formation of heme-protein cross-links and dimeric species.

Ferric leghemoglobin in the presence of H2O2 is known to give rise to protein radicals, at least one of which is centred on a tyrosine residue. These radicals are quenched by at least two processes. The first one involves an intramolecular heme-protein cross-link probably involving the tyrosine radical; this leads to the formation of a green compound with spectral characteristics differing markedly from those of ferryl and ferric leghemoglobin. This green compound cannot be reduced by dithionite or ascorbate, precluding any role for this species as an oxygen carrier. It exhibits modified EPR and pyridine haemochromogen spectra, indicating that alterations occur at the porphyrin macrocycle level. The additional compound previously described [Puppo, A., Monny, C. and Davies, M.J. (1993) Biochem. J. 289, 435-438] appears to be a mixture of ferry Lb and this green compound. The second quenching route results in the formation of intermolecular cross-links and hence dimeric forms of the protein. Ascorbate and glutathione inhibit both this intermolecular dimer formation and the formation of the intramolecular haem-protein cross-links and are likely to play a protective role in vivo.

Peroxidase content of soybean root nodules.

A peroxidase has been isolated from soybean nodules and its main characteristics have been determined. Its molecular weight (48 000) and spectral properties are similar to those of usual plant peroxidases. Its activity is comparable to that of low-efficiency plant peroxidases. The rate constant of the reaction with H2O2 is 3 x 10(5) M-1 x s-1. In this reaction, nodule peroxidase yields an oxidized intermediate analogous to the compound I species of peroxidases already studied. A comparison is made with the pseudoperoxidatic activity of soybean leghemoglobin components. Leghemoglobins a and c react with H2O2 with rate constants of 5 x 10(3) and 2.5 x 10(3) M-1 x s-1, respectively, yielding the leghemoglobin (IV) species. During these reactions leghemoglobins are inactivated.

Oxidative burst in alfalfa-Sinorhizobium meliloti symbiotic interaction.

Reactive oxygen species are produced as an early event in plant defense response against avirulent pathogens. We show here that alfalfa responds to infection with Sinorhizobium meliloti by production of superoxide and hydrogen peroxide. This similarity in the early response to infection by pathogenic and symbiotic bacteria addresses the question of which mechanism rhizobia use to counteract the plant defense response.

Relative survival in elderly European cancer patients: evidence for health care inequalities. The EUROCARE Working Group.

This paper examines the survival of elderly European cancer patients, on the basis of the EUROCARE II results. Using Hakulinen and Abeywickrama's method, the relative survival rates at 1 and 5 years from diagnosis were computed by sex and quinquennial age group for the elderly (65-99 years old). Age-standardised rates for the whole elderly group were also calculated. The analysis covered: all malignancies combined, stomach, colon, rectum, pancreas, lung, melanoma, bladder, kidney and non-Hodgkin's lymphomas for both sexes; prostate and larynx for men; and breast, ovary, uterine cervix and corpus for women. Data relating to 701521 cancer patients came from 44 population-based cancer registries in 16 European countries. The relative risks of death (RRs) of older patients (65-99) with respect to middle-aged adults (55-64) were computed by sex and country, for all malignancies only. The most prominent finding was the decrease in survival rates with increasing age for almost all cancer sites. The age-curves of survival rates at 1 year from diagnosis usually had a steeper slope than those at 5 years, particularly in women. This suggests that disease stage at presentation plays an important role in determining survival, particularly in the elderly. Thus, all factors which influence timing diagnosis in the elderly and cause a delay in tumour detection, such as psycho-social factors, access to care, co-morbidities and other clinical features affecting performance status, are very important predictors of prognosis. Very large geographic variations in relative survival rates were found among European countries. The ordering of countries was similar for almost all cancer sites. Western and Central Europe generally had the best survival, followed by Northern countries and by Southern ones (the latter with survival around the European average: 39% in men, 47% in women). The UK had survival rates unexpectedly lower than rates of nearest nations, often below the European average. Eastern countries usually had the lowest rates. In the very elderly patients (over 85 years), an apparent rise in the survival rates was noted, particularly at 5 years from diagnosis and in men. This 'too good' survival is unlikely to be due to real better prognosis, but rather to a selection bias. Countries with this unusual rise are also those registering a high proportion of DCO cases (those cases retrieved by death certificate only) (around 10%) or DCO unavailable. Another 'natural' bias has also to be taken into account: in elderly patients with a very bad prognosis, who are often suffering from other serious co-morbid conditions, cancer diagnoses could be under-notified and not reach at all the data sources commonly monitored by cancer registries.

Uptake of iron by symbiosomes and bacteroids from soybean nodules.

Symbiosomes and bacteroids isolated from soybean nodules are able to take up the iron-citrate complex. The kinetics are characterized by initial high rates of iron internalization, and ATPase inhibitors significantly lower the uptake. This is consistent with an energy-dependent process on both membranes, although the involvement of a simultaneous facilitated diffusion can not be completely ruled out. Citrate alone is poorly absorbed by symbiosomes; this uptake is greatly enhanced by addition of iron. Iron-citrate was found both in the nodule cytosol and in the bacteroids. These results provide the first experimental evidence for the existence, at least in young nodules, of an important iron trafficking system from the plant host cell to the microsymbiont, through the peribacteroid membrane.

How is leghemoglobin involved in peribacteroid membrane degradation during nodule senescence?

An increase in the rate of succinate and glutamate uptake by isolated symbiosomes from French bean nodules was observed in the presence of iron plus H2O2. The lipid bilayer, and not proteins involved in transport, seems to be the major target of radical attack. Leghemoglobin in the presence of a 6-fold excess of H2O2 (where heme breakdown and iron release occurred) provoked also an increase in peribacteroid membrane permeability. In contrast, this hemoprotein in the presence of a 2-fold excess of H2O2 (where a protein radical was generated) was without effect. We suggest that in vivo the release of heme iron may constitute the major process concerning the involvement of leghemoglobin in the degradation of the peribacteroid membrane during nodule senescence.

Direct detection of radicals in intact soybean nodules: presence of nitric oxide-leghemoglobin complexes.

Electron paramagnetic resonance spectroscopy has been employed to examine the nature of the metal ions and radicals present in intact root nodules of soybean plants grown in the absence of nitrate. The spectra obtained from nodules of different ages using this non-invasive technique show dramatic differences, suggesting that there are both qualitative and quantitative changes in the metal ion and radical species present. A major component of the spectra obtained from young nodules is assigned to a complex (Lb-NO) of nitric oxide (NO.) with the heme protein leghemoglobin (Lb). This Lb-NO species, which has not been previously detected in intact root nodules of plants grown in the absence of nitrate, is thought to be formed by reaction of nitric oxide with iron(II) leghemoglobin. The nitric oxide may be generated from arginine via a nitric oxide synthase-like activity present in the nodules of the soybean plants, in a manner analogous to that recently described for Lupinus albus. This Lb-NO complex is present at lower concentrations in older nodules, and is almost completely absent from senescent nodules. Exposure of young and mature nodules to oxidant stress, in the form of hydrogen peroxide, results in changes in the EPR spectra, with the loss of the signals from the Lb-NO complex and appearance of absorptions similar to those from untreated senescent nodules. These results suggest that there are characteristic changes in both the metal ion complexes and radicals present in intact root nodules of different ages, and support the theory that nitric oxide and other radicals play a significant role in determining the nitrogen fixing activity of root nodules; the modulatory activity of NO. may involve regulation of gene activity.

Prostate cancer in Italy before and during the 'PSA era': survival trend and prognostic determinants.

The aim of the study was to investigate the variations in prostate cancer prognosis during a period of major diagnostic change, such as the introduction of the prostate-specific antigen (PSA) test. Data were provided by 14 Italian cancer registries (CRs). Incidence and follow-up information was collected for patients diagnosed from 1978 to 1994. Relative survival was computed taking into account incidence period, age, tumour stage and grade at diagnosis. A multivariate analysis was carried out to evaluate the independent simultaneous effect on survival of some prognostic determinants. A large geographical variability was observed: in 1993-1994 Italian survival rates ranged from 76% to 52%, with a north-south gradient. A striking prognostic improvement (up to +27 percentage points) between the late 1980s and the early 1990s occurred in almost all CRs, particularly with regard to younger patients. Multivariate analysis showed a strong influence of incidence period on survival, also after correction by tumour stage. The slowdown of metastatic cancers suggests that the survival improvement could be due both to the introduction of an effective opportunistic screening and to a quantitative change in the application of clinical treatment, even if the effect of the lead-time bias phenomenon has to be taken into account.

Absence of synproportionation between oxy and ferryl leghemoglobin. off.

The synproportionation reaction between ferryl leghemoglobin and oxyleghemoglobin does not occur, at least under conditions where this process could be clearly demonstrated with myoglobin and hemoglobin. In contrast, a cross synproportionation can occur between oxyleghemoglobin and ferryl myoglobin or between ferryl leghemoglobin and oxymyoglobin. The non-exposure, at the surface of the leghemoglobin molecule, of the nearest tyrosine residue to the heme group could explain this behaviour. Thus leghemoglobin per se does not appear to be able to act as an antioxidant in removing H2O2 by synproportionation. However, in the presence of ascorbate and/or glutathione which can reduce ferryl leghemoglobin, this hemoprotein could act as an H2O2-removing antioxidant, in a process similar to that described for myoglobin. This could also explain why, despite the absence of synproportionation, ferryl leghemoglobin is not detected in nodule extracts.

Characterisation of a cDNA encoding gamma-glutamylcysteine synthetase in Medicago truncatula.

A gamma-ECS cDNA from Medicago truncatula was isolated using an Arabidopsis thaliana cDNA as probe. The analysis of the amino acid sequence deduced from this cDNA revealed 80% identity with the gamma-ECS from A. thaliana and Brassica juncea and suggested a plastidial localisation for the enzyme. Gamma-ECS activity and high level of GSH were detected in the gamma-ECS-deficient E. coli strain expressing a fusion protein containing the M. truncatula gamma-ECS protein. Southern blot analysis suggests that gamma-ECS is encoded by a small multigenic family in M. truncatula and shows that homologous genes are present in two other leguminous plants, Medicago sativa and Pisum sativum. Gamma-ECS gene expression was analysed by Northern blot in seedlings, plantlets and mature plants.

The reactivity of ascorbate with different redox states of leghaemoglobin.

Ascorbate has been previously shown to reduce the short-lived and reactive ferryl [Fe(IV) = O] states of myoglobin and haemoglobin. In this study it is shown that ascorbate is also able to reduce the otherwise long-lived and stable ferryl species [Lb(IV)] formed by the reaction of ferric or ferrous soybean leghaemoglobin with H2O2. The conversion of the ferryl species to ferric Lb, which appears to be moderately fast, is followed by a slow conversion of ferric Lb to oxyLb, by way of dexoy ferrous Lb. No reaction between ascorbate and oxyLb has been observed, in contrast to the previously reported pro-oxidant effect of ascorbate on oxymyoglobin. The addition of ascorbate prior to H2O2 to ferric Lb also prevents the formation of an additional species (which is not observed with myoglobin or haemoglobin); the optical spectra and migration on isoelectric focusing gels of this compound differ markedly from that of Lb(IV). When added after H2O2, ascorbate is able to reduce slowly this additional compound to oxyLb. These reactions are likely to occur in vivo as high ascorbate concentrations have been detected in soybean nodules. The reduction of Lb(IV) to ferric Lb is accompanied by oxidation of ascorbate, which has been detected by loss of the parent molecule in optical experiments and by the direct detection of ascorbate radicals by electron paramagnetic resonance (EPR) spectroscopy.(ABSTRACT TRUNCATED AT 250 WORDS)