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

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

Hereditary pancreatitis in children: surgical implications with special regard to genetic background.

PURPOSE: Hereditary pancreatitis (HP) is the primary etiology of chronic pancreatitis during childhood, progressing through recurrent episodes of acute pancreatitis and finally leading to pancreatic insufficiencies. Hereditary pancreatitis is because of mutations of the cationic trypsinogen (PRSS1) gene. Some other genes, such as SPINK1 or CFTR, have been associated with familial idiopathic chronic pancreatitis. The aim of our study was to clearly define diagnostic and therapeutic strategies for HP patients, through an analysis of our study group and a review of the literature. METHODS: All children admitted from 1995 to 2007 with a final diagnosis of hereditary pancreatitis were restrospectively included in the study. We analyzed all medical records with special attention given to cases involving genetic screening (PRSS1, SPINK1, and CFTR genes). RESULTS: Ten children were included. Eight had HP with PRSS1 mutation, 2 of them without a familial history of chronic pancreatitis. The 2 others patients had SPINK1 mutations. Three HP patients were operated on for acute complications of pancreatitis and are well with a mean follow-up of 5.5 years. No patient had pancreatic insufficiencies or weight loss. CONCLUSIONS: Hereditary pancreatitis is associated with severe pancreatitis, with a greater risk of developing pancreatic cancer. It must therefore be diagnosed correctly and treated to prevent its considerable complications

Staged gastroschisis closure using Alexis wound retractor: first experiences

INTRODUCTION: The aim of this study is to analyze the effectiveness of an Alexis wound retractor (AWR) device for staged gastroschisis closures. PATIENTS AND METHODS: AWR device was used to cover unreduced viscera of a gastroschisis when primary abdominal wall closure was not convenient. The eviscerated organs were covered with one of the two spring-loaded rings of the AWR inserted underneath the abdominal wall. Gradual reduction was guaranteed through careful traction on the external ring. We retrospectively analyzed the prenatal, post-natal and operative data of the first patients treated with AWR and report their post-operative outcomes. RESULTS: The AWR device was used for staged closure in eight cases. Complete reduction and fascial closure were performed at a median of 3.5 ± 1.6 days. Ventilatory support was necessary for 4.0 ± 3 days and full parenteral feeds for 7.5 ± 6.1 days after fascial closure. Median full enteral feeding was observed at 18 ± 12.5 days after closure allowing discharge in a median period of 30.5 ± 15.6 days after closure. CONCLUSION: The AWR device is not only a safe and efficient silo for a progressive reduction of severe gastroschisis, but also an interesting tool for continuous stretching leading to an increase of the peritoneal cavity volume, enhancing the equalizing of the viscero-abdominal disproportion

A new surgical approach to improve gene transfer in liver using lentiviral vectors.

PURPOSE: Metabolic inherited liver diseases are attractive targets for gene therapy. Recombinant lentiviruses are very powerful viral vectors able to infect nonmitotic cells. We wanted to develop a new surgical approach to improve gene transfer in adult liver using low viral doses. MATERIALS AND METHODS: Adult rats were injected with 2.108 infectious particles of lentiviral vectors encoding the green fluorescent protein marker gene under control of a liver-specific promoter transthyretin. In the control group (n = 5), gene delivery was performed by inflow intraportal injection. In the surgical group (n = 5), liver was completely excluded from systemic circulation before viral injection in infrahepatic vena cava with high pressure. RESULTS: At day 9, transduction efficiency was 14.35% in the surgical group 3 and 0.39% in the control group (P = .016). At month 2, the number of transduced hepatocytes decreased in the most part of rats, except in half of rats in the surgical group. Antibodies against green fluorescent protein were detected in all rats at month 2, except one in the surgical group. CONCLUSIONS: We developed a new surgical approach allowing an efficient transduction of hepatocytes in adult rats using lentivirus at low viral doses. We have now to control the immune response to permit long-term expression of transgene

The impact of anorectal malformations on anorectal function and social integration in adulthood: report from a national database.

AIM: The impact of anorectal malformation (ARM) on bowel function and social, educational and occupational end-points was investigated in adult patients entered on a national database. METHOD: Data from a national database of adult patients operated on between 1962 and 1999 for ARM were analysed. The database Malformations Ano-rectales et Pelviennes rares (MAREP) was part of a common information system, CEMARA, on rare congenital disorders. A self-administered questionnaire regarding bowel function, academic qualifications, employment and family status was mailed to patients. The type of ARM, subsequent follow-up and management including surgical interventions were retrospectively retrieved from medical records. RESULTS: Of 210 adult patients on the registry since 2008, 68 were included in this study. Only three (8.5%) had had regular follow-up. All reported some disturbance in bowel function. The fertility rate of 1.5 children per woman did not differ from the general population. CONCLUSION: Anorectal malformation ARM often leads to suboptimal bowel function in adulthood. This has an impact on social integration

Tolerance and efficacy of preventive gastrostomy feeding in pediatric oncology

BackgroundMalnutrition in pediatric oncology remains underestimated, although having a negative impact on outcome. Enteral nutrition (EN) using percutaneous endoscopic gastrostomy (PEG) may prevent or reverse malnutrition consequences. We aimed to evaluate both efficacy and safety of early EN during tumors treatment in children. Procedures Medical records of pediatric patients having a PEG tube inserted between 1995 and 2009 were retrospectively reviewed. We compared type and incidence of complications in Group 1, including 74 patients suffering from cancer, and control Group 2, including 57 patients with neurological impairment. Efficacy of EN was evaluated through nutritional parameters [Z-scores weight for height (W/H) and height for age (H/A)], post-operative complications and relapse rates. Statistical significance was set for P < 0.05. Results PEG tolerance was similar in both groups, as shown by comparable complication rates (62% vs. 76%, NS). EN allowed improvement or stabilization of Z-score W/H in 76% of oncologic patients. The final height loss was lower (−0.5 vs. −1.2 SD of Z-scores H/A) when EN was started at the beginning of the oncologic treatment. In bone tumors, EN prevented weight loss during chemotherapy, and tended to lessen surgical complications, relapses and deaths. Conclusions Early gastrostomy feeding represents a relatively safe way to prevent malnutrition in children with cancer, and might play a role in bone tumors oncological outcome. Further prospective studies are needed to confirm these results and assess the impact of EN and PEG on quality of life

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

The Microbiology of Community-acquired Peritonitis in Children

BACKGROUND: microbiologic data are lacking regarding pediatric community-acquired peritonitis (CAP). METHODS: we conducted a 2-year retrospective single center study. Consecutive children undergoing CAP surgery were included. Microbiology and antimicrobial susceptibility of peritoneal isolates were analyzed. RESULTS: a total of 70 children from 3 months to 14 years of age were included. A total of 123 bacterial isolates were analyzed. Escherichia coli was the predominant aerobic organism (51% of isolates); 54.8% were susceptible to amoxicillin whereas 90.3% were susceptible to amoxicillin-clavulanate. Anaerobes accounted for 29% of isolates, and 94.3% of strains were susceptible to amoxicillin-clavulanate and 68.5% were susceptible to clindamycin. Pseudomonas aeruginosa was present in 6% of isolates and in 10% of children. The presence of E. coli resistant to amoxicillin or to amoxicillin-clavulanate was the only independent risk factor associated with postoperative peritonitis. CONCLUSION: microbiology of pediatric CAP is similar to adult CAP with a predominancy of E. coli and anaerobes. P. aeruginosa in peritoneal samples had no apparent influence on the outcome