Automated Item Generation: impact of item variants on performance and standard setting.

BACKGROUND: Automated Item Generation (AIG) uses computer software to create multiple items from a single question model. There is currently a lack of data looking at whether item variants to a single question result in differences in student performance or human-derived standard setting. The purpose of this study was to use 50 Multiple Choice Questions (MCQs) as models to create four distinct tests which would be standard set and given to final year UK medical students, and then to compare the performance and standard setting data for each. METHODS: Pre-existing questions from the UK Medical Schools Council (MSC) Assessment Alliance item bank, created using traditional item writing techniques, were used to generate four 'isomorphic' 50-item MCQ tests using AIG software. Isomorphic questions use the same question template with minor alterations to test the same learning outcome. All UK medical schools were invited to deliver one of the four papers as an online formative assessment for their final year students. Each test was standard set using a modified Angoff method. Thematic analysis was conducted for item variants with high and low levels of variance in facility (for student performance) and average scores (for standard setting). RESULTS: Two thousand two hundred eighteen students from 12 UK medical schools participated, with each school using one of the four papers. The average facility of the four papers ranged from 0.55-0.61, and the cut score ranged from 0.58-0.61. Twenty item models had a facility difference > 0.15 and 10 item models had a difference in standard setting of > 0.1. Variation in parameters that could alter clinical reasoning strategies had the greatest impact on item facility. CONCLUSIONS: Item facility varied to a greater extent than the standard set. This difference may relate to variants causing greater disruption of clinical reasoning strategies in novice learners compared to experts, but is confounded by the possibility that the performance differences may be explained at school level and therefore warrants further study.

X-ray micro-computed-tomography in pediatric surgery: a new tool for studying embryos.

PURPOSE: The embryology of common congenital malformations is discussed controversially. Studies are hampered by a shortage of study material and techniques which require partial or complete preparation and therewith destruction of embryos. X-ray micro-computed-tomography (µCT) is a technical opportunity keeping the embryos intact. Thus, the aim of this study was to assess the applicability of µCT in embryonic research compared to the anatomical information obtained by scanning electron microscopy (SEM). METHODS: Chicken, rat, mouse and sheep embryos, processed either for SEM studies or as whole embryos, were imaged in three-dimensional (3D) using µCT. The obtained two-dimensional (2D) digital datasets were volume rendered by tomographic reconstruction software and studied using analysis software. RESULTS: All embryos were µCT scanned without technical problems. The quality of the µCT images (image contrast, anatomical details) was excellent, but varied depending on age and species studied. µCT imaging allowed a more comprehensive anatomical/morphological analysis but showed less surface details compared to SEM. CONCLUSION: µCT is a technique suitable and innovative for pediatric surgical research, which allows detailed evaluation of entire embryos without time- and specimen-consuming micro-dissection. Samples prepared for SEM can be used for µCT and vice versa.

Intrarenal B Cell Cytokines Promote Transplant Fibrosis and Tubular Atrophy.

Renal transplantation is the optimum treatment for end-stage renal failure. B cells have been identified in chronic allograft damage (CAD) and associated with the development of tertiary lymphoid tissue within the human renal allograft. We performed renal transplantation in mice to model CAD and identified B cells forming tertiary lymphoid tissue with germinal centers. Intra-allograft B220(+) B cells comprised of IgM(high) CD23(-) B cells, IgM(lo) CD23(+) B cells, and IgM(lo) CD23(-) B cells with elevated expression of CD86. Depletion of B cells with anti-CD20 was associated with an improvement in CAD but only when administered after transplantation and not before. Isolated intra-allograft B cells were cultured and shown to synthesize multiple cytokines, the most abundant of these were GRO-α (CXCL1), RANTES (CCL5), IL-6 and MCP-1 (CCL2). Tubular loss was observed with T cell accumulation within the allograft and development of interstitial fibrosis, whilst type III collagen deposition was observed in areas of F4/80(+) macrophages and PDGFR-ß(+) and transgelin(+) fibroblasts, all of which were reduced by B cell depletion. We have shown that intra-allograft B cells are key mediators of CAD. B cells possibly contribute to CAD by intra-allograft secretion of cytokines and chemokines.

Foetal hepatocyte transplantation in a vascularized AV-Loop transplantation model in the rat.

The use of foetal liver cells (FLC) in the context of hepatic tissue engineering might permit efficient in vitro expansion and cryopreservation in a cell bank. A prerequisite for successful application of bioartificial liver tissue is sufficient initial vascularization. In this study, we evaluated the transplantation of fibrin gel-immobilized FLC in a vascularized arterio-veno-venous (AV)-loop model. FLC were isolated from embryonic/foetal (ED 16) rat livers and were enriched by using magnetic cell sorting (MACS). After cryopreservation, FLC were labelled by pkh-26. Cells were transplanted in a fibrin matrix into a subcutaneous chamber containing a microsurgically created AV-loop in the femoral region of the recipient rat. The chambers were explanted after 14 days. Subcutaneous implants without an AV-loop and cell-free implants served as controls. Fluorescence microscopy of the constructs was used to identify pkh-26(+)- donor cells. Characterization was performed by RT-PCR and immunhistology (IH) for CK-18 and CD31. Transplantation of FLC using the AV-loop permitted a neo-tissue formation in the fibrin matrix. A high-density vascularization was observed in the AV-loop constructs as shown by CD31 IH. Viable foetal donor cells were detected which expressed CK-18. FLC can be successfully used for heterotopic transplantation. Fibrin matrix permits rapid blood vessel ingrowth from the AV-loop and supports engraftment of FLC. It is therefore an appropriate environment for hepatocyte transplantation in combination with microsurgical vascularization strategies. Transplantation of fibrin gel-immobilized FLC may be a promising approach for the development of highly vascularized in vivo tissue-engineering-based liver support systems.

[Fetoscopic surgery as prenatal intervention for isolated congenital diaphragmatic hernia]. / Die fetoskopische Chirurgie als Möglichkeit der pränatalen Intervention bei der isolierten angeborenen Zwerchfellhernie.

Congenital diaphragmatic hernia (CDH) occurs sporadically with an incidence of 1:2,500 live births. Despite the progress in neonatal intensive care, CDH remains associated with a mortality of at least 30 % in isolated cases. The in essence surgically correctable defect of the diaphragm enables the prenatal herniation of abdominal organs into the thoracic cavity. The resulting abnormal development of the airways and pulmonary vessels causes neonatal respiratory insufficiency and persistent pulmonary hypertension. The condition can be diagnosed prenatally and the degree of pulmonary hypoplasia, which determines the postnatal course, can be measured to make an -individual prognosis. In severely affected patients, prenatal surgery may improve neonatal outcome by reversing pulmonary hypoplasia. This is currently implemented by percutaneous fetoscopic endoluminal tracheal occlusion (FETO) to trigger fetal lung growth. Although there are no maternal complications, preterm rupture of the membranes remains the major drawback of the procedure (20 % < 34 weeks). However, as compared to historical controls of a similar severity, survival as well as early neonatal morbidity are significantly improved by FETO. As a consequence, a multicentre randomised-controlled trial in fetuses with moderate hypoplasia on FETO compared to expectant management has been started ( www.totaltrial.eu). Primary outcome measure is survival without chronic lung disease (i. e., with-out bronchopulmonary dysplasia). A trial in severely affected -fetuses with survival as main outcome is currently under review by ethics committee. A standardised neonatal management enables optimal treatment and multicentre compatibility. It remains to be proven if fetoscopic surgery can maintain a solid position in the prenatal treatment of CDH to improve both mortality and morbidity of the affected children.

Submucosal plexus of dilatated gut disappears after ligation in chicken embryos: preliminary results.

INTRODUCTION: Dilatation and impaired function of the gut is a condition often seen in newborns with bowel obstruction caused by intestinal atresia. In a previous experimental study in chicken embryos, we established a model to study ultrastructural changes during the development of the enteric nervous system after small bowel ligation. The aim of this study is to investigate the changes of the enteric nervous system (ENS) after gut ligation. METHODS: 56 chicken embryos were investigated. In the operation group fertilized eggs and the allantoic membrane were opened and the small bowel was ligated on embryonal day (ED) 11. The controls were sham-operated. The gut was prepared and harvested for analysis on ED 11, 12, 13, 14, 15, 16, 17 and 18. Silver staining or staining of the specimens for acetylcholinesterase (AchE) was performed. RESULTS: A marked dilatation of the bowel was observed three days after operation (ED 14). The submucosal (PSM) and myenteric plexus (PM) appeared normal at this time, however silver staining showed rarification of the neuronal axonal network between the myenteric and submucosal plexus. Later, on ED 16 an additional rarification of the submucosal plexus was also seen in the operation group using AchE staining, compared to the controls. DISCUSSION: The data suggest that distension of the gut hinders normal development of the ENS in the gut ligation model of chicken embryos. The changes were observed sequentially, starting with rarification of the axonal network between the PM and PSM. Future studies will be required to show whether the changes of the ENS are reversible.

Hepatocytic gene expression in cultured rat mesenchymal stem cells.

The origin of liver cells from distinct bone marrow stem cells, eg, hematopoietic stem cells or multipotent adult progenitor cells has been recently described using in vitro studies. Cell culture experiments revealed the key role of growth factors and the organ-specific environment for the induction of liver-specific genes. We investigated the in vitro potential of rat mesenchymal stem cells to differentiate into hepatocytic cells in cocultures with isolated rat liver cells. Rat mesenchymal stem cells (MSCs) propagated in culture, and transduced with green fluorescent protein (GFP) were cloned. Cells from selected clones were either cultured under liver-stimulating conditions, using serum free medium supplemented with HGF, EGF, SCF, and FGF-4 alone on fibronectin-coated surfaces, or cocultured with freshly isolated rat liver cells. Cocultured cells were harvested after two weeks and sorted into GFP-positive (GFP+) and GFP-negative (GFP-) cells. RT-PCR for liver specific markers CK-18 and albumin were performed on the different cell populations. After 2 weeks, the specified culture conditions led to the expression of albumin and CK-18 RNA in GFP-positive sorted MSCs from the cocultures, whereas MSCs cultured without liver cells did not express the studied genes. The results indicate, that when cocultured with liver cells MSCs from the bone marrow have the potential to differentiate toward hepatocytic cells in vitro. We conclude that MSC may possess an enhanced capacity to differentiate into functional liver cells. Additionally, environmental factors seem to be crucial for specific and directed differentiation.

Is there an optimal concentration of cotransplanted islets of Langerhans for stimulation of hepatocytes in three dimensional matrices?

BACKGROUND: Hepatocyte transplantation using three-dimensional matrices is under investigation as an alternative therapy for several liver diseases. For sufficient transplantation results hepatotrophic stimulation is necessary. We investigated the stimulatory effect of cotransplanted pancreatic islets in different ratios. METHODS: Lewis rats were used as donors and recipients. A portocaval shunt (group A) or sham operation (groups B-G) was performed 1 week before hepatocyte transplantation. Four polyvinyl-alcohol matrices each containing 1.25 x 10(7) hepatocytes (groups A and B) or 1.25 x 10(7) hepatocytes and 125 (C), 250 (D), 500 (E), or 750 (F) islets were implanted between small bowel mesenteric leaves. In group G, medium soaked matrices were implanted. One month after implantation, specimens were harvested and investigated using albumin-RNA in situ hybridization, and insulin, glucagon, and bromodesoxy uridine immunohistochemistry. The hepatocyte area was assessed using image analysis. RESULTS: Hepatocyte area and proliferation ratio increased depending on the number of cotransplanted islets with a peak at 40 islets per 1 million hepatocytes (group E). Cotransplantation of islets in higher concentrations did not further increase hepatocyte area or proliferation ratio. Hepatocytes in all groups expressed albumin RNA at normal transcription levels as compared to standard liver sections. Islets displayed insulin and glucagon in physiological distribution. DISCUSSION: Three-dimensional matrices provide a sufficient environment for transplanted hepatocytes and islets. The hepatotrophic effect of cotransplanted islets is comparable to portocaval shunting and has a saturation limit at 40 islets per 1 million hepatocytes. For further application of islet cotransplantation, this ratio seems to be preferable.

Hepatocyte transplantation using biodegradable matrices in ascorbic acid-deficient rats: comparison with heterotopically transplanted liver grafts.

BACKGROUND: Hepatocyte transplantation using polymeric matrices is under investigation as an alternative therapy for metabolic liver diseases. Long-term engraftment of hepatocytes in polymers has been demonstrated. However, the metabolic activity of hepatocytes in such devices has never been assessed in direct comparison with liver grafts. METHODS: Hepatocyte and partial liver transplantation were evaluated in the scurvy-prone osteogenic disorder Shionogi rat model. Biodegradable poly glycolic acid matrices seeded with hepatocytes equivalent to 20% of the recipient's liver mass, or 20% liver grafts were heterotopically transplanted into ascorbic acid- (AsA) deficient recipients. Recipients of cell-free matrices or AsA-deficient liver grafts served as controls. Recipients were set on AsA-free diet after transplantation. Plasma AsA levels, AsA concentrations in liver and adrenal gland tissue, and body weight ratios were assessed and H&E histology was performed. RESULTS: Recipients from the control groups showed symptoms of scurvy at 1 month after cessation of AsA supply. Hepatocyte transplantation and auxiliary liver transplantation prevented symptoms of scurvy and increased plasma and tissue AsA levels and body weight ratios. AsA levels in recipients of 20% liver grafts were comparable to normal control animals. CONCLUSIONS: Hepatocytes transplanted in polymeric matrices are able to compensate for liver-based metabolic deficiencies. Hepatocyte transplantation improves plasma AsA levels in AsA-deficient recipients. However, auxiliary liver grafts are superior to hepatocyte grafts in improving metabolic parameters. Further research work is needed to increase the efficiency of liver cell transplantation with regard to a clinical application.

Disseminated islands of gastric mucosa in jejunum and ileum detected by technetium-99m-pertechnetate scintigraphy.

Disseminated islands of gastric mucosa are very rare in the small intestine. The secretion of hydrochloric acid can lead to ulceration which results in gastrointestinal bleeding. It is often difficult to localize the focus in case of gastrointestinal blood loss especially in the small bowel. Technetium-99m-pertechnetate scintigraphy may be a helpful tool in detecting ectopic gastric mucosa. We report a case of a 21-mo-old boy with recurrent gastrointestinal bleeding. By using pertechnetate scintigraphy, extensive tracer accumulation in the jejunum and proximal ileum was detected. Histologically, multiple islands of ectopic gastric mucosa were found in about 50 excited mucosal and transmural biopsies. The unusual finding of disseminated accumulation of 99mTc-pertechnetate in the small intestine was the diagnostic clue for such a rare disease.

Inhibiting inflammatory cytokines.

Acute glomerulonephritis is a common cause of renal dysfunction and ultimately renal failure. The inflammation involved is a tightly regulated response with pro- and anti-inflammatory cytokines playing key roles. Interleukin-1 (IL-1) and tumor necrosis factor (TNF) are the principal pro-inflammatory cytokines produced by intrinsic cells and infiltrating leukocytes. IL-1 and TNF can be directly antagonized using IL-1 receptor antagonist (IL-1ra) or binding proteins such as soluble receptors or antibodies. Alternatively, cytokines with anti-inflammatory properties can be used to decrease IL-1 and TNF synthesis, increase the production of their natural antagonists and deactivate inflammatory cells such as macrophages. This review will focus on these anti-inflammatory cytokines, principally IL-4, IL-6, IL-10 and IL-13, and highlight recent research of their activities in existing models of renal disease. The results of these experiments offer a promising new avenue of treatment.

Priming of hepatocytes for cell culture by partial hepatectomy prior to cell isolation.

The combination of ex vivo gene transfer and a sufficient transplant model for hepatocytes may permit treatment of single enzyme-based metabolic liver diseases. Induction of replicative potential (priming) in hepatocyte cultures may enhance the efficiency of gene transfer under stable in vitro conditions. It is known that hepatocyte replication is increased in vivo after partial hepatectomy. We investigated the effect of partial hepatectomy prior to cell isolation on hepatocytes in vitro. Male Lewis rats served as donors. Hepatocytes were isolated by collagenase digestion from either intact livers or from livers 48 h after 70% hepatectomy (PH). Cells were seeded on collagen-coated culture dishes with hormone-supplemented culture media. Hepatocyte morphology, number, albumin secretion rate, and mono-ethyl-glycin-xylidid (MEGX)-biotransformation capacity were assessed on days 1, 3, and 5 in culture. PH significantly increased hepatocyte number and albumin secretion of cultured hepatocytes over the whole observation period. In contrast, MEGX-biotransformation capacity was significantly decreased. Morphology of cultured hepatocytes was not affected by PH prior to hepatocyte isolation. These results suggest a prolonged and complex response of hepatocytes to PH in vitro. Hepatocyte priming by PH is a promising approach toward stable cultures of proliferating hepatocytes and may provide a model for in vitro studies of hepatic regeneration mechanisms. Further research on hepatocyte priming toward an application in ex vivo gene transfer and hepatic tissue engineering seems justified.

Influence of pancreatic islets on growth and differentiation of hepatocytes in co-culture.

Improvement of cell culture conditions in hepatic tissue engineering may permit cell/tissue banking and the generation of liver tissue equivalents for transplantation. In these systems, continuous hepatotrophic stimulation is still necessary. We investigated the stimulatory effects of pancreatic islets on hepatocytes in co-culture and characterized the stimulatory mechanisms. Hepatocytes and pancreatic islets were harvested from Lewis rats. Cells were cultured on collagen dishes either with nonstimulated media (controls and co-cultures with low or high islet rate) or stimulated media (controls and co-cultures). To characterize stimulatory mechanisms, additional co-cultures with membrane separation, with antiinsulin, antiglucagon, and with both antibodies were examined. Hepatocyte numbers, albumin secretion rate by enzyme-linked immunoadsorbent assay, and monoethylglycinxylidid biotransformation values by fluorescence polarization immunoassay were assessed. A radioimmunoassay measured insulin and glucagon concentrations. In groups with nonstimulated media, cell number was higher in co-cultures with low islet rate, and albumin secretion rate was increased in co-cultures with high islet rate compared to controls. MEGX biotransformation was decreased in co-cultures. In groups with stimulated media, co-culture had no impact on cell number or albumin secretion rate. Hepatocyte numbers and albumin secretion rates were not changed in co-cultures after membrane separation. Islet effects on hepatocytes were reduced in co-cultures with antiinsulin, antiglucagon, or both antibodies. Pancreatic islets provide stimulation for hepatocytes in vitro. Islet effects were mediated by soluble factors, and are dependent on insulin and glucagon. These results permit further investigations towards three-dimensional transplantable hepatocyte-islet devices for continuous in vitro and in vivo stimulation.

Interaction of hepatocytes and pancreatic islets cotransplanted in polymeric matrices.

Heterotopic hepatocyte transplantation (HcTx) in polymeric matrices may become an alternative to liver transplantation for metabolic disorders. Hepatotrophic stimulation by means of a portocaval shunt operation is an established, but invasive, procedure used to optimize hepatocyte engraftment in matrices. We evaluated hepatocyte and pancreatic islet cotransplantation (ICT) as an alternative noninvasive approach to hepatotrophic stimulation. Lewis rats served as donors and recipients. Hepatocytes and islets were isolated using collagenase digestion and seeded into polyvinylalcohol matrices. HcTx and ICT were compared with HcTx plus portocaval shunt and HcTx without stimulation. Matrices were investigated at 1, 3, and 6 months after implantation: the test methods applied were trichrome staining, PAS, immunohistochemistry for insulin, glucagon and incorporated BrdU, and in situ hybridization for albumin RNA. Hepatocytes expressed albumin RNA and formed conglomerates without atypias in all animals. ICT and portocaval shunting increased the number of hepatocytes and BrdU uptake. Alpha cells migrated into the islet-surrounding hepatocytes, whereas beta cells remained immobile. It is concluded that ICT and portocaval shunting supported engraftment of hepatocytes in polymeric matrices equally well. ICT did not interfere with recipient glucose metabolism and did not induce hyperproliferative premalignant foci within the transplanted hepatocytes. The technique is an attractive approach to hepatotrophic stimulation of bioartificial liver equivalents.

Bleeding esophageal duplication detected in utero.

Peptic complications in duplications of the alimentary tract may occur when the duplication is lined by gastric mucosa. These complications commonly develop in the first year of life. We report a case of a bleeding esophageal duplication in a fetus.

Pulmonary echinococcosis (hydatidosis) in children: results of surgical treatment.

From 1986-1996, 33 children with 49 pulmonary hydatid cysts underwent surgical treatment in Vienna and Istanbul. Cysts were unilateral in 28 and bilateral in 5 cases; unruptured cysts (URC) were diagnosed in 19 patients, and 14 children presented with ruptured cysts (RC). Ten patients had cysts in other organs (liver, spleen, central nervous system) in addition to pulmonary cysts. Diagnosis was primarily based on chest X-ray and computed tomography scan. In Austrian children, a new combination of serological tests was used successfully (71% positive). The standard surgical procedure was cystotomy followed by capitonnage. The main postoperative complications were fever and wound infection. There were two recurrences after a mean follow-up of 4.8 years, and one patient died because of multiple organ involvement. We conclude that the therapy of choice in pediatric pulmonary hydatidosis is complete surgical elimination of the cyst by cystotomy and capitonnage, whereas more extended resections should be avoided. Ideally, benzimidazole treatment should be combined with surgery. New serological tests can improve diagnostic accuracy.

Targeting genetically modified macrophages to the glomerulus.

Macrophages are key players in the development of the majority of renal diseases and are therefore ideal cellular vectors for site specifically targeting gene therapy to inflamed glomeruli. Macrophages can be genetically modified using viral vectors ex vivo then re-introduced into the body where they can home to the diseased site. This review summarises current experience in efficiently targeting modified macrophages to the inflamed glomerulus focussing on the factors controlling macrophage localisation, macrophage gene transfer methods, in vivo gene delivery and results of recent investigations using modified macrophage gene therapy for glomerular disease.

New approaches to modify glomerular inflammation.

Glomerulonephritis remains the leading cause of end-stage renal failure and treatments for these conditions remain non-specific and with significant side effects. The cellular and molecular basis of acute and chronic inflammation is increasingly understood and the work in a number of animal models of nephritis demonstrates the potential of specific molecular interventions. These include preventing the migration of inflammatory cells by inhibiting the effects of chemokines or blocking endothelial/leucocyte adhesion interactions. Within damaged tissue it is possible to decrease the activity of pro-inflammatory cytokines, such as interleukin-1 (IL-1) and tumour necrosis factor (TNF) by using their natural antagonists, namely interleukin-1 receptor antagonist (IL-1ra) and soluble TNF receptors. In addition the behaviour of macrophages can be altered by the effects of anti-inflammatory cytokines including interleukin-4 (IL-4), interleukin-13 (IL-13), interleukin-10 (IL-10), interleukin-6 (IL-6) and transforming growth factor-beta (TGF-beta). By deactivating the inflammatory response of macrophages these cytokines can favour resolution of disease. The ability to use these approaches in clinical practice remains elusive, however the prospect of using gene transfer technology to deliver anti-inflammatory factors directly to the site of inflammation and our increasing understanding of the complexity of the control of inflammation bring such therapies closer.

Current concepts in the embryology of anorectal malformations.

Today, the normal and abnormal development of the hindgut is still a matter of speculation. However, owing to recent studies in appropriate animal models, most embryological events that finally lead to abnormal hindgut development are better known than in the past: (1) The process of maldevelopment starts early in the embryo. (2) The cloacal membrane always is too short in its dorsal part. Thus the dorsal cloaca is missing too. (3) As a result, the hindgut remains attached to the sinus urogenitalis, thus forming the recto-urethral fistula. In the past, an impaired process of septation was believed to be the main cause of abnormal hindgut development. In contrast to this, our results indicate that the development of the septum is more passive than active. Further results of our studies in normal and abnormal development indicate that (1) the embryonic cloaca never passes through a stage that is similar to any form of anorectal malformation in neonates, including the so-called "cloacas" in females, and (2) to explain abnormal development, studies in abnormal embryos are mandatory.

Embryology of congenital diaphragmatic hernia.

It is still generally believed that the defect in congenital diaphragmatic hernia results from failure of the so-called pleuroperitoneal canals (PPCs) to close at the end of the embryonic period (8th gestational week). Furthermore, it is assumed that gut could enter the thoracic cavity through this defect, causing compression and finally hypoplasia of the lung. However, this sequence of embryological events has never been studied, and many details even of normal diaphragmatic development are still unknown. Using scanning electron microscopy and a new animal model of congenital diaphragmatic hernia (CDH), the nitrofen rat model, the normal embryology of the diaphragm was reinvestigated and, for the first time, the crucial developmental steps of congenital diaphragmatic hernia formation were studied. The basic results were: (1) In normal development, the PPCs are never wide enough to allow herniation of gut loops. (2) The formation of the defect happens in an early embryonic period. (3) The early ingrowth of liver through the defect is of major importance for the formation of CDH. In another set of experiments, the nitrofen rat model of congenital diaphragmatic hernias was used to study the cellular mechanisms involved during epithelial and mesenchymal growth and differentiation in normal and in abnormal lungs. These results, combined with selected culture techniques (eg, branching morphogenesis and epithelio-mesenchymal interaction) probably open new ways to a better understanding of the mechanisms that finally lead to an abnormal lung in CDH.