In vivo retrovirus-mediated gene transfer into lamb liver.

TOPIC: Highly efficient retrovirus-mediated gene transfer into hepatocytes in vivo has been previously reported in the rat. Before considering human applications of these techniques in the treatment of inherited liver diseases, it was necessary to document its efficiency in a large animal model. Lamb was choosen because the liver was similar to human liver regarding size and anatomy. MATERIALS AND METHODS: To induce hepatocyte division which is necessary for infection with retroviral particles, animals were subjected to a left hepatectomy. Kinetics of liver regeneration were assessed on sequential liver biopsies after partial hepatectomy in order to provide an evaluation of the peak of maximal liver regeneration in a first animal group. Recombinant retroviruses encoding a reporter gene (E. coli beta galactosidase) were then perfused through the portal vein of the regenerating liver in a second animal group. RESULTS: The more intense liver regeneration occurred from one to 6 days after partial hepatectomy, with the highest thymidine kinase rate and MIB-1 antibody staining on the second day. The proportion of genetically modified lamb hepatocytes expressing the reporter gene was less than 1%, despite the use of higher titers of retroviral particles than those described in previous reports. CONCLUSION: The results obtained in rodent livers with this in vivo gene transfer methodology cannot currently be scaled up in a large ruminant model. The efficacy of vectors has to be tested in other large mammals before planning gene therapy trials for the treatment of inherited liver diseases

Sperm morphology, swimming velocity, and longevity in the house sparrow Passer domesticus

Sperm competition exerts strong selection on males to produce spermatozoa with an optimal morphology that maximizes their fertilization success. Long sperm were first suggested to be favored because they should swim faster. However, studies that investigated the relationship between sperm length and sperm competitive ability or sperm swimming velocity yielded contradictory results. More recently, ratios of the different sections of a spermatozoon (the head, midpiece, and flagellum) were suggested to be more crucial in determining swimming velocity. Additionally, sperm ability to remain and survive in the female storage organs may also influence fertilization success, so that optimal sperm morphology may rather maximize sperm longevity than velocity. In this study, we investigated how sperm morphology is related to sperm velocity and sperm longevity in the house sparrow Passer domesticus. Sperm velocity was found to be correlated with head/flagellum ratio. Sperm with small heads relative to their flagellum showed higher swimming velocity. Additionally, shorter sperm were found to live longer. Finally, we found sperm morphological traits to vary substantially within males and the head/flagellum ratio to be unrelated to total sperm length. We discuss the hypothesis that the substantial within-male variation in sperm morphology reflects a male strategy to produce a diversity of sperm from long, fast-swimming to short, long-living sperm to maximize their fertilization success in a context of sperm competitio

In vivo retroviral-mediated transfer of a marker-gene in ornithine transcarbamylase-deficient Spf(ash) mice.

Gene therapy is a new therapeutic approach for inherited metabolic hepatopathies. The authors studied the potential application of such a strategy to the correction of ornithine transcarbamylase (OTC) deficiency by in vivo protocol of retroviral-mediated gene transfer to the liver. A partial hepatectomy was followed (24 to 48 hours later) by asanguinous perfusion of the regenerating liver with beta-galactosidase (beta-gal) recombinant retrovirus. This protocol allowed beta-gal gene transfer in normal C57B6 mice liver with 60 +/- 52 positive cells per square centimeter. This proportion never exceeded 20 cells per square centimeter in OTC-deficient spf(ash) mice. The high mortality rate for spf(ash) mice was explained by an important sensitivity of those mice to the protein catabolism rather than by technical difficulties during intraportal perfusion. This first in vivo retroviral-mediated gene transfer study in animals with a life-threatening metabolic inherited hepatopathy showed that, despite efficiency of gene therapy in normal animal models, several experimental difficulties should be overcome before human application of this protocol is considered

The challenge of measuring quality of life in children with Hirschsprung's disease or anorectal malformation

PURPOSE: The aim of the present study was to assess, after adaptation to French, the only specific quality of life (QoL) instrument for children with Hirschsprung\u27s disease or anorectal malformation, the Hirschsprung\u27s disease/Anorectal Malformation Quality of Life questionnaire (HAQL), in order to get a standardized QoL evaluation instrument that could further be used to help health care improvement. METHODS: The study was conducted in three teaching hospitals, including the French reference center for anorectal and pelvic malformations. After adaptation to French, QoL questionnaires were sent to the children and proxies. The questionnaire was mailed to 280 families. Psychometrics properties of the questionnaires (validity and reliability) were analysed from 120 proxy and 96 child questionnaires. RESULTS: The HAQL with the original structure was not acceptable. Exploratory steps led to a clinically pertinent structure that had acceptable fit and good validity and reliability properties. The final structure pools physical symptoms (continence, discomfort) and psychosocial dimensions (general well-being, social and emotional functioning) of QoL. CONCLUSION: The final structure, despite the disadvantage of being a new structure, allows assessment of QoL in this population and has the advantage of being shorter and validated on the clinical postoperative questionnaire from the Krickenbeck international consensus

Consecuencias de la colonización y de la modernidad sobre la cultura y los modos de vida en América Latina y en Colombia

Después de la llegada de los europeos, Colombia nunca fue la misma. A través de este ensayo, voy a mostrar y analizar los cambios en la forma de vida de los indígenas de Colombia, principalmente aquellos relacionados con su cultura. ¿Quiénes eran los pueblos indígenas antes de la colonización? ¿Quiénes son los pueblos indígenas hoy? ¿Cuál es el impacto de estos cambios y de la modernidad sobre la economía del país? Tantas preguntas que voy intentar responder. Por fin, lo que llamamos modernidad tiene un impacto sobre la vida de los colombianos. ¿Cuáles son los impactos y cómo añadir a eso los efectos del cambio climático

Learning Laparoscopic Skills: Observation or Practice?

OBJECTIVE: The aim of this study was to assess the respective roles of observation and direct practice in the retention of laparoscopic skills. MATERIALS AND METHODS: Eighteen fifth-year medical students were included in a two-session laparoscopic learning course. During the first session, each participant was given four tasks to complete from the "Basic skills" and "Essential tasks" modules of the Simbionix LAP Mentor™, and another four tasks for observation only. During the second session, each participant completed all eight tasks. Performance evaluation was assessed using the objective structured assessment of technical skills (OSATS) global rating scale and LAP Mentor metrics. RESULTS: The mean OSATS score during the first session (S1) was 16.7 ± 3.2. This increased by 34% during the second session (S2), reaching 21.8 ± 2.6 in the group of former observer students (S2O, P < .0001), and by 56% (25.1 ± 1.9) in the group of former practicing students (S2A, P < .0001). The analysis of LAP Mentor metrics showed that 14 of 28 parameters (50%) improved in the S2A group compared to S1, whereas only 25% of the parameters improved in the S2O group, the difference being significant (P = .048). In both groups, the more complex the task, the more the number of improved parameters decreased. CONCLUSIONS: Although simple observation of laparoscopic skills improved further performance, direct practice on the virtual reality trainer ensured more effective training. This work therefore advocates incorporating personal training on simulators into residents\u27 surgical curricula

Skills improvement after observation or direct practice of a simulated laparoscopic intervention

BACKGROUND: Laparoscopic skills are more and more often being learned on simulators. PURPOSE: To assess the respective roles of observation and direct practice in the retention of laparoscopic procedural skills. BASIC PROCEDURES: Twelve surgical residents were included in a two-session laparoscopic training course. During the first session (S1), one participant completed a cholecystectomy on the Simbionix LAPMentor™ and then observed his colleague carrying out a total hysterectomy and vice versa. During the second session (S2), each participant completed both interventions. Skills evaluation was assessed using the Objective Structured Assessment of Technical Skills (OSATS) global rating scale and LAPMentor™ metrics. MAIN FINDINGS: Mean OSATS score during the first session was 19.3±5.1, and increased by 37% in the group of former observer students (S2O, P=0.003), and by 54% in the group of former practising students (S2A, P=0.001). Self- and peer-grading results were concordant with the supervisor\u27s evaluation. Detailed analysis of LAPMentor™ metrics showed a trend toward more parameters being improved in group S2A as compared to group S2O on both interventions. The most significant improvements concerned the time of completion for the hysterectomy and the efficiency of cautery for the cholecystectomy. CONCLUSIONS: Observation of laparoscopic skills still allows for surgical improvement, but direct practice on a virtual reality trainer provides better results. Self- and peer-grading were concordant with the supervisor\u27s evaluation. This work may advocate the integration of both personal training on simulators and surgical observation into residents\u27 surgical curricula

Selection and validation of reference genes for quantitative RT-PCR expression studies of the non-model crop Musa

Gene expression analysis by reverse transcriptase real-time or quantitative polymerase chain reaction (RT-qPCR) is becoming widely used for nonmodel plant species. Given the high sensitivity of this method, normalization using multiple housekeeping or reference genes is critical, and careful selection of these reference genes is one of the most important steps to obtain reliable results. In this study, reference genes commonly used for other plant species were investigated to identify genes displaying highly uniform expression patterns in different varieties, tissues, developmental stages, fungal infection, and osmotic stress conditions for the non-model crop Musa (banana and plantains). The expression stability of six candidate reference genes was tested on six different sample sets, and the results were analyzed using the publicly available algorithms geNorm and NormFinder. Our results show that variety, plant material, primer set, and gene identity can all influence the robustness and outcome of RT-qPCR analysis. In the case of Musa, a combination of three reference genes (EF1, TUB and ACT) can be used for normalization of gene expression data from greenhouse leaf samples. In the case of shoot meristem cultures, numerous combinations can be used because the investigated reference genes exhibited limited variability. In contrast, variability in expression of the reference genes was much larger among leaf samples from plants grown in vitro, for which the best combination of reference genes (L2 and ACT genes) is still suboptimal. Overall, our data confirm that the stability of candidate reference gene

Factors influencing immune response after in vivo retrovirus-mediated gene transfer to the liver.

BACKGROUND: Highly efficient retrovirus-mediated gene transfer into hepatocytes in vivo triggers an immune response directed against transduced hepatocytes. This effect may be due either to spreading of retroviral vectors in the blood stream with subsequent infection of antigen presenting cells (APCs) or to cross-presentation of the transgene product present as a contaminant in the viral stock. In order to decrease immune response, we evaluated the effect of asanguineous perfusion of the liver as well as purification of the viral stock on long-term transduction of hepatocytes using the nls-lacZ marker gene. METHODS: Animals were divided in four groups. In group 1, the viral supernatant was perfused in the regenerating liver after complete vascular exclusion of the organ. In group 2, using the same strategy, animals received retroviral supernatant that was passed through a beta-galactosidase affinity column to reduce beta-galactosidase contamination. In two control groups (respectively groups 3 and 4) the corresponding viral supernatants were delivered via peripheral injection. RESULTS: In group 1, 23.1% of animals had no immune response 2 months after gene delivery vs. 33.4% in group 2, 4.3% in control group 3, and 0% in control group 4. Statistical analysis of the results demonstrated that only the difference between groups 2 and 3 was statistically significant. This indicated that both asanguineous perfusion together with passage through an affinity column were required to decrease significantly immune response. CONCLUSIONS: Our present results suggest that both supernatant contamination and viral spreading contribute to immune response after retrovirus-mediated gene delivery to the liver