Lung size and liver herniation predict need for extracorporeal membrane oxygenation but not pulmonary hypertension in isolated congenital diaphragmatic hernia: systematic review and meta-analysis.

OBJECTIVES: To identify antenatal predictors of persistent pulmonary hypertension (PPH) and the need for extracorporeal membrane oxygenation (ECMO) in fetuses with congenital diaphragmatic hernia (CDH). METHODS: We performed a systematic literature review on antenatal diagnostic tests in fetuses with isolated CDH. The primary outcomes assessed were PPH within 28 days of age and the need for ECMO. Quality of studies was assessed with the QUADAS-2 tool. Meta-analysis was performed when at least three studies reported on the same test. Sensitivity analysis was performed according to prenatal management of CDH (tracheal occlusion vs expectant management). RESULTS: Thirty-eight studies met the inclusion criteria. Fifteen reported on the incidence of PPH only, 19 on the need for ECMO only and four reported on both outcomes. The general quality of the studies was moderate; most studies were retrospective (61%) and single-center series (92%). One study included only fetuses undergoing tracheal occlusion, 22 included only fetuses managed expectantly in utero and 15 included both populations. We could not identify antenatal predictors of PPH. The need for ECMO was predicted by parameters indicative of lung size: lung-to-head ratio (LHR) (relative risk (RR) for LHR < 1, 1.65 (95% CI, 1.27-2.14)) and observed/expected LHR (standardized mean difference (SMD), -0.70 (95% CI, -0.98 to -0.42)) measured by ultrasound and observed/expected total lung volume (SMD, -1.00 (95% CI, -1.52 to -0.48)) measured by magnetic resonance imaging. Liver herniation was also associated with an increased risk of need for ECMO (RR, 3.04 (95% CI, 2.23-4.14)). These results were confirmed by a sensitivity analysis of studies that included only expectantly managed cases. Data on vascular assessment for the prediction of PPH could not be pooled as most of the parameters were evaluated in a single series or in different series by the same principal investigator. CONCLUSIONS: In fetuses with CDH, lung size and liver herniation predict the need for ECMO, however a predictor for PPH is still lacking. Further studies aimed at diagnosing impaired vascular development in utero should therefore be undertaken. Copyright © 2016 ISUOG. Published by John Wiley & Sons Ltd.

Clinical prognostic value of combined analysis of Aldh1, Survivin, and EpCAM expression in colorectal cancer.

BACKGROUND: Tumour aggressiveness might be related to the degree of main cancer hallmark acquirement of tumour cells, reflected by expression levels of specific biomarkers. We investigated the expression of Aldh1, Survivin, and EpCAM, together reflecting main cancer hallmarks, in relation to clinical outcome of colorectal cancer (CRC) patients. METHODS: Immunohistochemistry was performed using a tumour tissue microarray of TNM (Tumour, Node, Metastasis)-stage I-IV CRC tissues. Single-marker expression or their combination was assessed for associations with the clinical outcome of CRC patients (N=309). RESULTS: Increased expression of Aldh1 or Survivin, or decreased expression of EpCAM was each associated with poor clinical outcome, and was therefore identified as clinically unfavourable expression. Analyses of the combination of all three markers showed worse clinical outcome, specifically in colon cancer patients, with an increasing number of markers showing unfavourable expression. Hazard ratios ranged up to 8.3 for overall survival (P<0.001), 36.6 for disease-specific survival (P<0.001), and 27.1 for distant recurrence-free survival (P<0.001). CONCLUSIONS: Our data identified combined expression levels of Aldh1, Survivin, and EpCAM as strong independent prognostic factors, with high hazard ratios, for survival and tumour recurrence in colon cancer patients, and therefore reflect tumour aggressiveness.

Validation of 70-gene prognosis signature in node-negative breast cancer.

PURPOSE: The 70-gene prognosis signature (van't Veer et al., Nature 415(6871):530-536, 2002) may improve the selection of lymph node-negative breast cancer patients for adjuvant systemic therapy. Optimal validation of prognostic classifiers is of great importance and we therefore wished to evaluate the prognostic value of the 70-gene prognosis signature in a series of relatively recently diagnosed lymph node negative breast cancer patients. METHODS: We evaluated the 70-gene prognosis signature in an independent representative series of patients with invasive breast cancer (N = 123; <55 years; pT1-2N0; diagnosed between 1996 and 1999; median follow-up 5.8 years) by classifying these patients as having a good or poor prognosis signature. In addition, we updated the follow-up of the node-negative patients of the previously published validation-series (Van de Vijver et al., N Engl J Med 347(25):1999-2009, 2002; N = 151; median follow-up 10.2 years). The prognostic value of the 70-gene prognosis signature was compared with that of four commonly used clinicopathological risk indexes. The endpoints were distant metastasis (as first event) free percentage (DMFP) and overall survival (OS). RESULTS: The 5-year OS was 82 +/- 5% in poor (48%) and 97 +/- 2% in good prognosis signature (52%) patients (HR 3.4; 95% CI 1.2-9.6; P = 0.021). The 5-years DMFP was 78 +/- 6% in poor and 98 +/- 2% in good prognosis signature patients (HR 5.7; 95% CI 1.6-20; P = 0.007). In the updated series (N = 151; 60% poor vs. 40% good), the 10-year OS was 51 +/- 5% and 94 +/- 3% (HR 10.7; 95% CI 3.9-30; P < 0.01), respectively. The DMFP was 50 +/- 6% in poor and 86 +/- 5% in good prognosis signature patients (HR 5.5; 95% CI 2.5-12; P < 0.01). In multivariate analysis, the prognosis signature was a strong independent prognostic factor in both series, outperforming the clinicopathological risk indexes. CONCLUSION: The 70-gene prognosis signature is also an independent prognostic factor in node-negative breast cancer patients for women diagnosed in recent years.

Nuclear distribution of the Ki-67 antigen during the cell cycle: comparison with growth fraction in human breast cancer cells.

It has been claimed that the commercially available Ki-67 monoclonal antibody recognizes a nuclear antigen which is solely expressed in cycling cells. Therefore, at present, Ki-67 is increasingly used as a tool in evaluating growth fractions (GFs) of human tumors. Here we describe specific patterns in the expression and topological distribution of this antigen during the cell cycle in MCF-7 human breast cancer cells. Our results support earlier findings that the antigen belongs to a class of antigens associated with the structural organization of meta- and anaphase chromosomes, and proteins located near the cortical regions of prenucleolar bodies and nucleoli. Using 5'-bromodeoxyuridine-labeling technology, we show that the expression may be undetectably low at the onset of DNA replication. Comparison of Ki-67 fractions (KFs) and GFs as estimated from continuous 5'-bromodeoxyuridine-labeling curves revealed that KF was invariably higher than GF: in exponentially growing cov362.c14 human ovary cancer cells, KF was only 3.5% higher than GF; in MCF-7 cells, 11.3 +/- 4.6%. In MCF-7 cultures either growing under suboptimal conditions or treated with 10(-6) M tamoxifen, the difference was more pronounced. Furthermore, we evaluated the decrease of Ki-67-positive cells in nutritionally deprived and cell cycle-specific, drug-treated cultures. Since the results indicate that nonproliferating cells may retain the antigen for a considerable period of time, KF may not always be a reliable indicator of GF.

Bcl-2 and Bax proteins are present in interphase nuclei of mammalian cells.

The Bcl-2 family of proteins comprises both cell death inhibiting and cell death promoting members, generally believed to be cytoplasmic and predominantly membrane-associated. Like Bcl-2, many Bcl-2-related proteins contain a C-terminal membrane insertion domain and much research is aimed at evaluating the functional role of their localization to the outer membranes of mitochondria, the endoplasmic reticulum, and perinuclear membranes. However, confocal fluorescence microscopy of human breast cancer cells and rat colon cancer cells immunostained with commercial antibodies raised against different epitopes of the anti-apoptotic Bcl-2 and the pro-apoptotic Bax protein revealed that these proteins are not only present in the cellular cytoplasm, but also within interphase nuclei. This was confirmed by Western blot analysis of isolated nuclei. In human cells, certain epitopes of Bcl-2, but not of Bax, were also found to be associated with mitotic chromatin. Anti-estrogen treatment of human breast cancer cells or transfection with antisense bcl-2 led to a reduction in both cytoplasmic and nuclear Bcl-2. Transfection of human bcl-2 and bax into rat cells resulted in cytoplasmic and nuclear Bcl-2 and Bax. This data seems in line with increasing evidence that the role of the Bcl-2 family of proteins should be extended to activities inside the nuclear compartment.

Branching and differentiation defects in pulmonary epithelium with elevated Gata6 expression.

The transcription factor GATA6 is expressed in the fetal pulmonary epithelium of the developing mouse lung and loss of function studies strongly suggested that it is required for proper branching morphogenesis and epithelial differentiation. We have further investigated the role of GATA6 in this process by utilizing a pulmonary epithelium specific promoter to maintain high levels of GATA6 protein during fetal lung development. Transgenic mice expressing Gata6 cDNA under the control of the human Surfactant Protein-C (SP-C) promoter were generated and their lungs were analyzed during fetal stages. Transgenic lungs exhibit branching defects as early as embryonic day (E) 14.5 and molecular analysis just before birth (E18.5) shows a lack of distal epithelium differentiation whereas proximal epithelium is unaffected. Electron microscopic analysis and glycogen staining confirm the lack of differentiation to mature Type II cells. Thus, elevated levels of GATA6 protein affect early lung development and in analogy to other GATA factors in other tissues, GATA6 also plays a crucial role in the terminal differentiation in this case of the distal pulmonary epithelium.

The role of various Bcl-2 domains in the anti-proliferative effect and modulation of cellular glutathione levels: a prominent role for the BH4 domain.

Reduced cell proliferation and increased levels of cellular glutathione (GSH) are characteristic for cells that overexpress the anti-apoptotic Bcl-2 protein. We investigated the influence of various Bcl-2 domains on both these characteristics. Rat CC531 colorectal cancer cells were stably transfected with the human bcl-2 gene (CCbcl2 cells) or with bcl-2 gene constructs missing a coding sequence for a func-tional domain, BH1 (CCDeltaBH1 cells), BH3 (CCDeltaBH3 cells), BH4 (CCDeltaBH4 cells) or the transmembrane region (CCDeltaTM cells). We measured GSH levels in exponentially and confluent growing bcl-2-transfected cell populations. The fraction of S-phase cells during exponential growth was significantly reduced in CCbcl2, CCDeltaBH1, CCDeltaBH3, and CCDeltaTM cells compared with parental CC531, neo-transfected CC531 and CCDeltaBH4 cells. GSH levels in these bcl-2 transfectants were significantly higher than in the parental line measured at 50% confluence; at 100% confluence they reached a similar level as found in parental cells. Independently from the presence of BH1, BH3 or TM domains, overexpression of Bcl-2 reduces cellular proliferation under conditions of increased GSH levels. This apparent link is lost in CCDeltaBH4 cells; these cells are not reduced in cellular proliferation and harbour significantly higher GSH levels than found in the other transfectants. Studies on the subcellular localization revealed an extremely low expression of the Bcl-2 protein lacking the N-terminal BH4 domain in nuclear fractions. Nuclear translocation of Bcl-2 requires the presence of the BH4 domain and seems prominent in reducing cellular proliferation.

A new method to detect apoptosis in paraffin sections: in situ end-labeling of fragmented DNA.

Apoptosis (programmed cell death) can be difficult to detect in routine histological sections. Since extensive DNA fragmentation is an important characteristic of this process, visualization of DNA breaks could greatly facilitate the identification of apoptotic cells. We describe a new staining method for formalin-fixed, paraffin-embedded tissue sections that involves an in situ end-labeling (ISEL) procedure. After protease treatment to permeate the tissue sections, biotinylated nucleotides are in situ incorporated into DNA breaks by polymerase and subsequently stained with DAB via peroxidase-conjugated avidin. Staining of cells with the morphological characteristics of apoptosis was demonstrated in tissues known to exhibit programmed cell death, i.e., prostate and uterus after castration, tumors, lymph node follicles, and embryos. Apoptotic cells could be discriminated morphologically from areas of labeled necrotic cells, in which DNA degradation also occurs. Because apoptosis is relatively easily recognized in H&E-stained sections of involuting prostates of castrated rats, we used this model system to validate the ISEL method for the quantification of apoptotic cells. A high correlation was found between the fractions of ISEL-labeled cells and the fractions of apoptotic cells that were morphologically determined in adjacent sections. We conclude that ISEL is a useful technique for quantification of apoptosis in paraffin sections, especially for those tissues in which morphological determination is difficult. Furthermore, this new staining method enables the use of automated image cytometry for evaluating apoptosis.

Routine formaldehyde fixation irreversibly reduces immunoreactivity of Bcl-2 in the nuclear compartment of breast cancer cells, but not in the cytoplasm.

Bcl-2 and Bax belong to a family of proteins involved in apoptosis regulation and are believed to reside in the cellular cytoplasm. The authors recently reported interphase nuclear localization of both proteins after immunofluorescence staining of formaldehyde- and methanol-fixed human and rodent cell monolayers. In addition, the authors' data confirmed earlier reports on Bcl-2 immunoreactivity of mitotic chromosomes in human cells. In their experience, nuclear or mitotic staining of Bcl-2, in contrast with cytoplasmic Bcl-2 immunoreactivity, is rarely observed in formaldehyde-fixed, paraffin-embedded breast cancer specimens. Therefore, the authors wondered if nuclear and mitotic Bcl-2 immunoreactivity could be irreversibly reduced by certain fixation procedures, including formaldehyde fixation. Here the authors investigated the effects of various routinely used fixation protocols and antigen retrieval techniques on Bcl-2 and Bax immunoreactivity in monolayers of MCF-7 human breast cancer cells. Whereas nuclear and mitotic immunoreactivity for Bcl-2 was clearly present after formaldehyde and methanol fixation, it was completely absent in cells fixed in acetone, methanol, or formaldehyde alone. In addition, it was found that in particular nuclear and mitotic Bcl-2, and to a lesser extent cytoplasmic Bcl-2 immunoreactivity, decreased after prolonged formaldehyde fixation, whereas Bax immunoreactivity diminished only slightly. Heat-mediated antigen retrieval after prolonged formaldehyde fixation elevated cytoplasmic, but not nuclear and mitotic, Bcl-2 immunoreactivity.

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.

Hormonal modulation of fetal pulmonary development: relevance for the fetus with diaphragmatic hernia.

Antenatal hormonal modulation of pulmonary growth has been successfully introduced in clinical practice to reduce the incidence of respiratory distress syndrome (RDS) of preterm born infants. However, a certain amount of reserve to repeat courses should be taken into account because of possible adverse effects of antenatal administration of glucocorticoids. Although in experimental animals thyroid hormones given alone were not shown to have stimulatory effects on pulmonary development, there was an apparent synergistic effect with corticosteroids. Yet, such effects have not been substantiated in clinical trials. Whereas in cases of congenital diaphragmatic hernia (CDH) in utero tracheal occlusion could stimulate fetal lung growth and modulation, the enhancement of type II cell differentiation is more likely to be achieved with antenatal exposure to hormonal therapies. However, there is still no firm scientific basis for either of these two treatment modalities in CDH. Yet, antenatal hormonal modulation is now soon to be tested in an extensive multi-center clinical trial. In this review, the current status of antenatal hormonal modulation of pulmonary growth will be described and its potential role in the treatment of CDH will be discussed.

Comparison of the effects of microwave heating and high pressure cooking for antigen retrieval of human and rat Bc1-2 protein in formaldehyde-fixed, paraffin-embedded sections.

Immunohistochemical detection of expression of the anti-apoptotic Bcl-2 protein is widely studied as a putative prognostic and predictive factor in various types of cancer. For that purpose, heating for 10 min by microwave (MW) up t o 100 degrees C in citrate buffer, pH 6.0, prior to immunostaining is often used to retrieve Bcl-2 antigens in archival formalin-fixed, paraffin-embedded tissue. We recently reported that Bcl-2 is not only a cytoplasmic protein, but that it is present also in interphase nuclei and that it strongly associates with mitotic chromosomes. Furthermore, we showed that binding of the monoclonal antibody (MAb) #124 with nuclear/chromosomal epitopes is diminished by formaldehyde-based fixatives and cannot be restored by MW treatment for 10 min. Here we report that prolonged MW heating or heating up to 130 degrees C in a high pressure cooker (HPC), despite improved cytoplasmic immunostaining, fails to retrieve nuclear/chromosomal Bcl-2 epitopes recognized by the MAb #124 in human tissues. In contrast, these procedures can retrieve nuclear/chromosomal Bcl-2 epitopes detected by polyclonal #15616E antibodies in rat tissues. The specificity of these epitopes was confirmed by Western blot analysis of tissues treated by MW heating or HPC.

[Diagnostic image (152). A man with a swelling in the groin. Malignant eccrine poroma]. / Diagnose in beeld (152). Een man met een gezwel in de lies. Maligne eccrien poroom.

A malignant eccrine poroma (porocarcinoma) was excised from the right groin of a 78-year-old man.

[Important prognostic significance of a sentinel-node biopsy in patients with malignant melanoma]. / Belangrijke prognostische betekenis van schildwachtklierbiopsie bij patiënten met maligne melanoom.

OBJECTIVE: To determine the prognostic significance of sentinel-node biopsy in patients with malignant melanoma (unlike the United States, a sentinel-node biopsy is still not routinely performed on melanoma patients in the Netherlands, as the outcomes of prospectively randomised clinical trials are being awaited). DESIGN: Retrospective. METHODS: Between 1996 and 2001 a sentinel-node biopsy and a re-excision of the scar of the diagnostic biopsy were performed on all melanoma patients who had a Breslow thickness > or = 1 mm or a Clark level > or = IV. At operation the sentinel node was identified with a gamma probe and patent blue. It was removed and sent for pathological investigation for the presence of melanoma cells. If the sentinel node was tumour positive, a dissection of the regional lymph-node basin was performed. Subsequently, these patients were put forward for the European Organisation for Research and Treatment of Cancer (EORTC) peginterferon alfa(2b) adjuvant treatment study. RESULTS: A sentinel-node biopsy was performed in 61 lymphnode basins in 57 patients (18 male and 39 female; median age: 45 years (range: 9-80)). The median Breslow thickness of the melanomas was 2.2 mm (range: 0.7-13 mm). In 10 of the 61 cases histological examination of the sentinel node demonstrated tumour cells. In 2 additional cases tumour cells were demonstrated only by immunohistochemical studies or complete dissection of the node. Eight regional lymph-node basins were dissected, two of which contained additional metastases. The median follow-up was 36 months (range: 1-68). During follow-up 12 of the 57 patients were found to have metastases, in 8 of these patients the sentinel-node biopsy contained tumour cells. The negative predictive value of a tumourless sentinel node with respect to the later occurrence of distant metastases was 92%. CONCLUSION: The patients with a tumour-positive sentinel node had a poorer prognosis with respect to distant metastases than patients with a tumour-negative node. This is the main reason for performing sentinel-node biopsy: to predict the prognosis of the disease. Therefore sentinel-node biopsy should be incorporated into the treatment of patients with malignant melanoma.

Postmortem biopsy to obtain lung tissue in congenital diaphragmatic hernia.

BACKGROUND: The accrual of human tissues from autopsies for diagnostic and translational research has decreased significantly over the last decades. OBJECTIVES: The objective of this study was to evaluate our experience with lung biopsy through a minithoracotomy as an alternative for obtaining postmortem tissue when full autopsy is refused in congenital diaphragmatic hernia (CDH) patients. METHODS: Within 2 h of death we routinely asked parents for permission to perform an autopsy. Starting in 2001, parents who refused autopsy were asked permission for a postmortem lung biopsy. Pathology autopsy and biopsy reports were compared to clinical records. RESULTS: Between 2001 and 2009, 46 patients died from CDH. Permission for autopsy was granted in 5 patients (11%). Of the remaining 41 patients, the parents of 15 (33%) agreed to postmortem lung biopsy. In all cases, additional findings were reported from the autopsy or biopsy, without changing the originally reported cause of death. In 1 case, we isolated fibroblasts from the lung biopsy using standardized cell culture techniques. Parents were able to take their child home with a minimal delay following biopsy. CONCLUSIONS: Parents refusing a full autopsy frequently agree to postmortem organ biopsy. This approach should therefore be considered as a valuable alternative, when permission for full autopsy is declined, for obtaining human tissues for both diagnostic and research purposes and is potentially applicable to other anomalies.

Etiological and pathogenic factors in congenital diaphragmatic hernia.

Congenital diaphragmatic hernia (CDH) is a congenital anomaly associated with an increased mortality and morbidity. In this article, we review the currently known etiological and pathogenic factors in CDH.

Genetics and developmental biology of oesophageal atresia and tracheo-oesophageal fistula: lessons from mice relevant for paediatric surgeons.

Oesophageal atresia and tracheo-oesophageal fistula are relatively frequently occurring foregut malformations of which the aetiology and pathogenesis are poorly understood. Recent results of molecular genetic studies, in particular the use of single and compound mutant mice, have yielded a tremendous increase in the understanding of the molecular mechanisms involved in normal and abnormal foregut morphogenesis. In the introduction of this paper, we review the very early stages of normal and abnormal embryology of the foregut derivatives and the separation of the foregut into a ventral respiratory part and a dorsal digestive part. After that, we describe the genes that have been demonstrated to play an important role in these processes.

Surgically induced cytokinetic responses in experimental rat mammary tumor models.

The effect of surgical removal of "primary" tumors on the cytokinetics of local tumor remnants, secondary implants, and metastases was investigated in three different rat tumor models in the Wag/Rij rat: a slow-growing (MCR83) and a fast-growing (EMR86) hormone-dependent mammary tumor and a rapidly, but autonomously growing carcinoma (MCR86). The latter two tumors had metastatic potential. Cell kinetic studies were done using in vivo labeling with 5'-bromodeoxyuridine (BrdUrd). Thirty-three hours after removal of a subcutaneous MCR83 flank tumor, secondary implants showed a significant (P less than 0.05) but transient increase in the BrdUrd labeling index (LI). A more rapid and prolonged increase, lasting for at least 7 days, was observed in EMR86 lymph node and lung metastases. In both models, no effect was observed after sham surgery (consisting of opening and closing of the skin under anesthesia). Removal of MCR86 tumors (growing in the hind leg muscle) also resulted in a rapid, transient LI increase in metastases. Continuous BrdUrd labeling experiments in this tumor model did not favor the hypothesis that the LI increase predominantly resulted from an increase in the growth fraction. Moreover, in this model, the effect was related to operation trauma. A similar increase in LI, although smaller than after tumor removal, was seen after major surgical trauma in MCR83 flank tumors. These results indicate that in the rat, tumor removal and/or major surgical trauma may modulate the cytokinetics of distant metastases significantly. A study of the systemic, possibly endocrine, factors involved in the growth-stimulating effect of surgical trauma in these rat tumor models may help to assess the clinical relevance of these findings for patients with breast cancer.

Dual-hit hypothesis explains pulmonary hypoplasia in the nitrofen model of congenital diaphragmatic hernia.

Pulmonary hypoplasia associated with congenital diaphragmatic hernia (CDH) remains a major therapeutic problem. Moreover, the pathogenesis of pulmonary hypoplasia in case of CDH is controversial. In particular, little is known about early lung development in this anomaly. To investigate lung development separate from diaphragm development we used an in vitro modification of the 2, 4-dichlorophenyl-p-nitrophenylether (Nitrofen) animal model for CDH. This enabled us to investigate the direct effects of Nitrofen on early lung development and branching morphogenesis in an organotypic explant system without the influence of impaired diaphragm development. Epithelial cell differentiation of the lung explants was assessed using surfactant protein-C and Clara cell secretory protein-10 mRNA expression as markers. Furthermore, cell proliferation and apoptosis were investigated. Our results indicate that Nitrofen negatively influences branching morphogenesis of the lung. Initial lung anlage formation is not affected. In addition, epithelial cell differentiation and cell proliferation are attenuated in lungs exposed to Nitrofen. These data indicate that Nitrofen interferes with early lung development before and separate from (aberrant) diaphragm development. Therefore, we postulate the dual-hit hypothesis, which explains pulmonary hypoplasia in CDH by two insults, one affecting both lungs before diaphragm development and one affecting the ipsilateral lung after defective diaphragm development.