F. Bargy

Engraftment of Autologous Retrovirally Transduced Hepatocytes after Intraportal Transplantation into Nonhuman Primates: Implication for ex Vivo Gene Therapy

The main impediment to effective ex vivo liver gene therapy of metabolic diseases is the lack of experimental work on large animals to resolve such important issues as effective gene delivery, cell-processing techniques, and the development of appropriate vectors. We have used a nonhuman primate, as a preclinical model, to analyze the limiting steps of this approach using recombinant retroviruses. Seven monkeys (Macaca fascicularis) underwent the complete protocol: their left liver lobe was resected, a catheter was placed in the inferior mesenteric vein and connected to an infusion chamber, and the hepatocytes were isolated, cultured, and transduced with a retroviral vector containing the beta-galactosidase gene. The hepatocytes were harvested and returned to the host via the infusion chamber. Biopsies were taken 4-40 days later. No animal was killed in the course of the experiments. They all tolerated the procedure well. We have developed and defined conditions that permit the proliferation and transduction of up to 90% of the plated hepatocytes. A significant proportion of genetically modified cells, representing up to 3% of the liver mass, were safely delivered to the liver via the chamber. Polymerase chain reaction analysis detected integrated viral DNA sequences and quantitative analysis of the in situ beta-Gal-expressing hepatocytes indicated that a significant amount of transduced hepatocytes, up to 2%, had become integrated into the liver and were functional. These results represent substantial advances in the development of the ex vivo approach and suggest that this approach is of clinical relevance for liver-directed gene therapy.

Fetal Lung Growth in Congenital Diaphragmatic Hernia

Background: In spite of significant therapeutic progress, the prognosis of congenital diaphragmatic hernia (CDH) remains pejorative in those forms in which the liver is herniated into the chest. The severity of this malformation relies on the pulmonary hypoplasia due to lung compression by the herniated viscera in the thoracic cavity, particularly the liver. This impaired growth concerns the whole pulmonary tissue, i.e. both the vessels and the alveoli. For the clinician, it is mandatory to know the evolution pattern of the lesions, to define the best time to treat them. Aim and Method: The aim of this work was to study the pulmonary lesions along the gestation in fetuses affected byCDH. This morphological study was carried out on 134 human fetuses aged from 22 to 40 weeks of gestation. Anatomical and histological analysis focused on lung weight, alveolar count and wall thickness of the distal vessels. Results: The results indicate that the pulmonary lesions worsen as the pregnancy continues, particularly beyond 30 weeks of gestation. Conclusion:Such an anatomical study should bring to the clinicians useful data to enhance the management of the patients.

Developmental Stages in the Rabbit Embryo: Guidelines to Choose an Appropriate Experimental Model

Researchers involved in the field of congenital malformations are often forced to work on an animal model. Both accurate description of its normal development and comparative staging with human development will be mandatory. To complete the lacking medical literature, we herein provide such data for the rabbit model. Sampled rabbit embryos were staged using the Carnegie criteria, in order first to determine if they were consistent with the rabbit developmental pattern, and second to compare this pattern with the human one. Our results show a suitable comparison of rabbits and humans in early developmental stages, except for the neural growth.

Anomalies of lateralization in man : a case of total situs inversus

Total or complete visceral situs inversus is the complete inversion of position of the thoracic and abdominal viscera. The aim of this study is to report a case of complete situs inversus and to review our knowledge of the anomalies of lateralization. A case of complete sinus inversus was discovered incidentally during anatomic dissection in a female subject aged 87 years. The thoracic and abdominal organs had a position symmetric with the normal. This was associated with a common mesentery and incomplete rotation of the colon, placing the cecum under the left lobe of the liver. These alimentary anomalies were discovered in adult life during a surgical operation for small intestinal occlusion, as evidenced by the abdominal scar and peritoneal adhesions. No cardiac, pulmonary, splenic or facial sinus anomalies were encountered. The incidence of complete situs inversus is estimated as 1/8000 in the general population. It may be isolated or associated with malformations, especially cardiac or alimentary. It may be discovered in infancy because of associated anomalies but often remains asymptomatic and discovered by chance in adult life. Complete situs inversus may form part of the multiple malformational syndromes such as that of Kartagener, with recessive autosomal transmission (complete situs inversus, bronchiectasis, chronic sinusitis, male infertility), which represents 20–25% of cases of complete situs inversus. In view of the frequency of this type of anomaly, a knowledge of anomalies of lateralization is essential in clinical practice.

Anatomic study of the umbilical vein and ductus venosus in human fetuses: ultrasound application in prenatal examination of left congenital diaphragmatic hernia

For clinicians it is very difficult to evaluate the prognosis of a left congenital diaphragmatic hernia (CDH) at prenatal ultrasound examination. Surgical studies show that the presence of a large part of the liver in the chest is a criterion of poor prognosis. However, ultrasonography encounters some difficulties in determining the precise position of the liver in the thoracic cavity. The aim of this anatomic study was to define the relationship between the position of the liver and the path of the ductus venosus and of the umbilical v., which are easily recognizable at prenatal sonography. Twenty dead fetuses were used for the study (12 with a left CDH and 8 without). All fetuses underwent radiographic assessment, anatomic dissection and cross-sectional study. The angle between the umbilical v. and the ductus venosus in different planes was measured. The more the liver was in the thorax, the greater was the angle between the ductus venosus and the sagittal plane, and the less the angle between the ductus venosus and the umbilical v. These angles can be easily measured by prenatal ultrasound examination of the fetus. Our findings suggest that it is now possible to offer the clinician a new and reliable way to determine the prognosis of a left CDH before birth.

Anatomical basis of surgical repair of hypospadias by spongioplasty.

Despite the numerous surgical procedures reported for hypospadias repair, little attention has been given to the precise assessment of penile anatomy in this malformation. In this study of 51 cases of hypospadias, we describe the particular anatomy of the corpus spongiosum encountered, highlighting its relationships with the other features observed in the different forms of this anomaly. It appears that the level where the corpus spongiosum diverges into two limbs particularly induces the degree of penile curvature that influences the severity in this malformation. We report a new technique of anatomic repair of the penis in hypospadias.

Outcome of 116 Moderate Renal Pelvis Dilatations at Prenatal Ultrasonography

To determine the incidence of urinary tract abnormalities detected in the presence of moderate fetal renal pelvis dilatation, we followed up pre- and postnatally 116 fetuses and children between 1985 and 1995. At prenatal ultrasound, 50 (43%) fetuses showed regressive dilatations, 57 (49%) a stable pattern, and 9 (8%) an evolutive pattern. In the presence of an evolutive dilatation, urinary tract obstruction was present in 8 cases. When a stable pattern was observed, i.e., a patent uropathy was present, surgical correction was performed in 27 of 53 (51%) cases. Regarding the postnatal evolution of 50 prenatal regressive moderate dilatations, we observed in 12 of 50 (24%) vesicoureteric reflux, of which 5 (10%) required surgical correction, and it is concluded that careful and extensive follow-up is mandatory.

Pelvic development in the rabbit embryo: implications in the organogenesis of bladder exstrophy

Current theses of the development of bladder exstrophy and its variants rely on defective evolution of the urinary tract and cloacal membrane. They do not account satisfactorily for the clinical features reported in children affected by exstrophy, especially the pelvic bone anomaly. We herein describe the normal development of the pelvic ring in the rabbit embryo and its chronological relationship with the lower urinary tract organogenesis. Our results suggest that these events are intricated and allow us to propose a novel mechanism to explain exstrophies.

Anatomical basis of a common embryological origin for epispadias and bladder or cloacal exstrophies

Epispadias, bladder exstrophy and its variants are in the first place usually considered as urological anomalies. Embryological theses and therapeutic approaches are mainly based upon this aspect. We challenge this point of view, in order to bring out a new axis of research about this still misknown pathologic field. A review of 16 cases of bladder exstrophy, 6 epispadias cases, and one cloacal exstrophy case, which had never been described before, revealed that the almost constant bony defect of the pelvic ring was linked with the severity of the visceral features, and with the continence status in epispadias cases. The commonly admitted theories about exstrophy development are based on a primary defect of the cloacal membrane. We suggest that the first anomaly could lie in a lack of rotation in the pelvic ring primordia.

In utero allotransplantation of retrovirally transduced fetal hepatocytes in primates: Feasibility and short-term follow-up

In utero allotransplantation of fetal hepatocytes into a preimmune fetus could be used in early treatment of many inherited hepatic metabolic diseases. This study was designed to assess the tolerance to hepatocyte transplantation and to test the feasability and toxicity of such an injection in a primate model. Fetal hepatocytes were obtained from two 120-day-old Macaca mulatta fetuses and cryopreserved. They were thawed, cultured in vitro, and transduced with a recombinant retrovirus expressing beta-galactosidase. Transduction efficiency was 75-85%. Three unrelated fetuses (90, 100, and 104 days old) were each given 1-2 x 10(7) transduced cells via the umbilical vein. This caused vasospasm and severe bradycardia. Two fetuses died in the 48 hours after transplantation; the third survived and was killed at the end of gestation. No evidence of the infused cells was found. Three fetuses (90 days old) were, therefore, given 3-4 10(7) hepatocytes by direct intrahepatic injection. All the fetuses survived without side effect. Donor cells were not apparent from histochemical staining and PCR reactions. There was no evidence of inflammatory reaction. These findings indicate that the protocole could be improved by increasing the number of transplanted cells and using specific hepatic promoters in the retroviral vectors to achieve an effective postnatal chimerism.