Expression of Eya1 and Six1 is decreased in distal airways of rats with experimental pulmonary hypoplasia

Background/Purpose

Pulmonary hypoplasia (PH) associated with congenital diaphragmatic hernia (CDH) represents one of the major challenges in neonatal intensive care. Eyes absent 1 (Eya1) and sine oculis homebox 1 (Six1) have been identified as essential components of the gene network that regulates foetal lung development. Eya1 and Six1 are expressed in distal epithelial tips of branching airways as well as in surrounding mesenchymal cells, highlighting their important role during branching morphogenesis. Lungs of Eya1^−/− and Six1^−/− knockouts display PH with reduced epithelial branching, appearing to be arrested in the pseudoglandular stage. We hypothesized that Eya1 and Six1 expression is decreased in branching airways of nitrofen-induced PH.

Methods

Time-mated rats received either nitrofen or vehicle on E9.5. Foetal lungs were dissected on E15.5 and divided into control and nitrofen groups, whereas lungs harvested on E18.5 were divided into controls, PH without CDH [PH(−)], and PH with CDH [PH(+)]. Pulmonary gene expression levels of Eya1 and Six1 were analyzed by quantitative real-time PCR. Immunofluorescence staining was performed to investigate Eya1 and Six1 protein expression and localization by confocal laser scanning microscopy (CLSM).

Results

Relative mRNA expression of Eya1 and Six1 was significantly decreased in PH(−) and PH(+) on E18.5 compared to controls. CLSM confirmed markedly diminished immunofluorescence of Eya1 and Six1 in distal airway epithelium as well as in surrounding mesenchymal cells of nitrofen-induced PH on E18.5 compared to controls.

Conclusions

Downregulation of Eya1 and Six1 gene expression in nitrofen-induced PH suggests that decreased Eya1 and Six1 expression during the late pseudoglandular stage may interfere with epithelial branching and distal airway maturation, thus resulting in PH.     

Prenatal retinoic acid increases lipofibroblast expression in hypoplastic rat lungs with experimental congenital diaphragmatic hernia

Background/purpose

Prenatal administration of all-trans retinoic acid (ATRA) has been shown to stimulate alveolarization in nitrofen-induced pulmonary hypoplasia (PH) associated with congenital diaphragmatic hernia (CDH). Lipid-containing interstitial lipofibroblasts (LIFs), characterized by adipocyte differentiation-related protein (ADRP), play a critical role in alveolar development by coordinating lipid homeostasis. Previous studies have demonstrated that ATRA positively affects LIF expression in developing lungs. We hypothesized that pulmonary LIF expression is increased after prenatal ATRA treatment in the nitrofen model of CDH-associated PH.

Methods

Timed-pregnant rats were treated with nitrofen or vehicle on E9.5, followed by injection of ATRA or placebo on E18.5, E19.5, and E20.5. Fetal lungs were dissected on E21.5 and divided into Control + Placebo, Control + ATRA, Nitrofen + Placebo, and Nitrofen + ATRA. Pulmonary gene expression levels of ADRP were analyzed by quantitative real-time polymerase chain reaction, and LIF expression was investigated by ADRP immunohistochemistry, oil-red-O-, and immunofluorescence-double-staining.

Results

Relative mRNA expression of pulmonary ADRP was significantly increased in Nitrofen + ATRA compared to Nitrofen + Placebo (0.31 ± 0.02 vs. 0.08 ± 0.01; P < 0.0001). ADRP immunoreactivity and oil-red-O-staining were markedly increased in alveolar interstitium of Nitrofen + ATRA compared to Nitrofen + Placebo. Immunofluorescence-double-staining confirmed markedly increased LIF expression in alveolar walls of Nitrofen + ATRA compared to Nitrofen + Placebo.

Conclusions

Increased LIF expression after prenatal treatment with ATRA in nitrofen-induced PH suggests that ATRA may have a therapeutic potential in attenuating CDH-associated PH by stimulating alveolar development.     

Interventionelle Therapie von tachykarden Herzrhythmusstörungen bei Kindern mit angeborenem Herzfehler

In children and adolescents with congenital heart disease (CHD) tachyarrhythmia occurs more frequently compared to patients with otherwise normal hearts. Arrhythmia substrates may be a natural part of certain congenital cardiac malformations or may result from long lasting myocardial deterioration as a result of CHD and/or cardiac surgery. Treatment of tachycardia is more frequently required even in early childhood, as the impact on quality of life, morbidity and mortality is higher due to an often reduced hemodynamic tolerance. Over the past 20 years interventional electrophysiology has been established as the therapy of choice for the majority of chronic or chronically recurrent tachycardia even in children with CHD. The success and risks of treatment are predominantly influenced by the individual expression of the cardiac anomaly and, if surgery has been performed, the highly variant postoperative anatomy. Introduction of 3D electroanatomical mapping systems together with modern cardiac imaging tools have significantly contributed to an improved understanding, particularly in postoperative tachycardia. Despite such progress, success rates are lower and recurrences are more frequent compared to patients without CHD. Complex and often multiple tachycardia courses account for the still limited performance as well as a frequently insufficient lesion formation with the use of radiofrequency current in the hypertrophic and fibrotic myocardium. Electrophysiology in children and adolescents, particularly if CHD is present, represents a highly specialized discipline requiring a high expertise in CHD, CHD surgery and cardiac electrophysiology and is ideally imbedded within an interdisciplinary cardiological and cardiosurgical setting.     

Disruption of THY-1 signaling in alveolar lipofibroblasts in experimentally induced congenital diaphragmatic hernia

Purpose

Pulmonary hypoplasia (PH), characterized by alveolar immaturity, remains the main cause of neonatal mortality and long-term morbidity in infants with congenital diaphragmatic hernia (CDH). Lipid-containing interstitial fibroblasts (LIFs) are critically important for normal alveolar development. Thymocyte antigen 1 (Thy-1) is a highly expressed cell-surface protein in this specific subset of lung fibroblasts, which plays a key role in fetal alveolarization by coordinating the differentiation and lipid homeostasis of alveolar LIFs. Thy-1 increases the lipid content of LIFs by upregulation of adipocyte differentiation-related protein (ADRP), a lipogenic molecular marker characterizing pulmonary LIFs. Thy-1 ^?/? mice further show impaired alveolar development with reduced proliferation of pulmonary LIFs, resulting in a PH-similar phenotype. We hypothesized that pulmonary Thy-1 signaling is disrupted in experimentally induced CDH, which may has an adverse effect on the lipid content of alveolar LIFs.

Methods

Timed-pregnant Sprague–Dawley rats were treated with either 100 mg nitrofen or vehicle on embryonic day 9.5 (E9.5). Fetuses were killed on E21.5, and lungs were divided into controls (n = 14) and CDH-associated PH (n = 14). Pulmonary gene expression levels of Thy-1 and ADRP were assessed by quantitative real-time PCR. ADRP immunohistochemistry and oil-red-O staining were used to localize alveolar LIF expression and lipid droplets. Immunofluorescence double staining for Thy-1 and oil-red-O was performed to evaluate Thy-1 expression and lipid content in alveolar LIFs.

Results

Radial alveolar count was significantly reduced in CDH-associated PH with significant downregulation of pulmonary Thy-1 and ADRP mRNA expression compared to controls. ADRP immunoreactivity and lipid droplets were markedly diminished in alveolar interstitial cells, which coincided with decreased alveolar LIF expression in CDH-associated PH compared to controls. Confocal laser scanning microscopy confirmed markedly decreased Thy-1 expression and lipid content in alveolar LIFs of CDH-associated PH compared to controls.

Conclusion

Our study provides strong evidence that disruption of pulmonary Thy-1 signaling results in reduced lipid droplets in alveolar LIFs and may thus contribute to PH in the nitrofen-induced CDH model. Treatment modalities aimed at increasing lipid content in alveolar LIFs may therefore have a therapeutic potential in attenuating CDH-associated PH.     

Substandard factors in perinatal care in The Netherlands: a regional audit of perinatal deaths

BACKGROUND: To determine: 1) whether substandard factors were present in cases of perinatal death, and to what extent another course of action might have resulted in a better outcome, and 2) whether there were differences in the frequency of substandard factors by level of care, particularly between midwives and gynecologists/obstetricians and between home and hospital births. METHODS: Population-based perinatal audit, with explicit evidence-based audit criteria. SETTING: The northern part of the province of South-Holland in The Netherlands. All levels of perinatal care (primary, secondary and tertiary care, and home and hospital births) were included. CASES: Three hundred and forty-two cases of perinatal mortality (24 weeks of pregnancy--28 days after birth). MAIN OUTCOME MEASURES: Scores by a Dutch and a European audit panel. Score 0: no substandard factors identified; score 1, 2 or 3: one or more substandard factors identified, which were unlikely (1), possibly (2) or probably (3) related to the perinatal death. RESULTS: In 25% of the perinatal deaths (95% Confidence Interval: 20-30%) a substandard factor was identified that according to the Dutch panel was possibly or probably related to the perinatal death. These were mainly maternal/social factors (10% of all perinatal deaths; most frequent substandard factor: smoking during pregnancy), and antenatal care factors (10% of all perinatal deaths; most frequent substandard factor: detection of intra-uterine growth retardation). We did not find statistically significant differences in scores between midwives and gynecologists/obstetricians or between home and hospital births. The European panel identified more substandard factors, but these were again equally distributed by level of care. CONCLUSIONS: Perinatal deaths might be partly preventable in The Netherlands. There is no evidence that the frequency of substandard factors is related to specific aspects of the perinatal care system in The Netherlands.     

Impact of the Site of Anastomosis after Oncologic Esophagectomy on Quality of Life — A Prospective,Longitudinal Outcome Study

Background For patients undergoing oncologic surgery, the quality of life (QoL) is generally accepted as an important outcome parameter in addition to long-term survival, mortality, and complication rates. Our study focused on outcome in terms of QoL in patients with esophageal cancer, comparing the sites of anastomosis (cervical versus thoracic anastomosis). Methods In a prospective longitudinal single-center study from 1998 to 2005, 105 patients underwent surgery for esophageal cancer. To assess QoL the EORTC-QLQ-C-30 and a tumor-specific module were administered before surgery, at discharge, and three, six, 12, and 24 months after surgery. Clinical data were collected prospectively and follow-up was performed every six months. Results The histological type was squamous cell carcinoma in 51.4% of the cases, adenocarcinoma in 41.9%, and some other type in 6.7%. There was no significant difference between cervical and thoracic anastomosis with regard to morbidity, mortality, and survival rates (30% five-year survival rate), whereas tumor stage was a significant (p < 0.001) prognostic factor. Most QoL scores dropped significantly below baseline in the early postoperative period. Even though they recovered slowly during the follow-up period, they never reached preoperative levels again. There was no statistically significant difference in any of the QoL scales between patients with a cervical or a thoracic anastomosis. Conclusions Esophageal resections are associated with significant deterioration of QoL, which persists during the follow-up period. The surgical technique and position of the esophagogastrostomy did not affect QoL deterioration. Jan-Hendrik Egberts and Bodo Schniewind contributed equally to this work.     

Kissing melanoma or kissing nevus of the penis?

Divided, or so-called kissing nevi of the penis, which are separated during embryogenesis, are very rare, with only seven studies reported to date. We present a case of a 30-year-old patient with a divided nevus located at the penis that rapidly changed in size and color within 4 months. These clinical features and the histopathologic appearance of both parts led to the diagnosis of malignant melanoma. Melanoma of the penis is very rare, and the prognosis is poor. This could be the first case of a "kissing melanoma" of the penis reported in published studies.     

Human neuromelanin induces neuroinflammation and neurodegeneration in the rat substantia nigra: implications for Parkinson's disease

Parkinson's disease (PD) is a common neurodegenerative disorder characterized by a selective loss of dopaminergic neurons in the substantia nigra (SN). It has been suggested that microglial inflammation augments the progression of PD. Neuromelanin (NM), a complex polymer pigment found in catecholaminergic neurons, has sparked interest because of the suggestion that NM is involved in cell death in Parkinson's disease, possibly via microglia activation. To further investigate the possible role of NM in the pathogenesis of PD, we conducted in vivo experiments to find out whether microglial cells become activated after injection of human neuromelanin (NM) into (1) the cerebral cortex or (2) the substantia nigra to monitor in this PD-relevant model both microglial activation and possible neurodegeneration. In this study, adult male Wistar rats received an intracerebral injection of either NM, bacterial lipopolysaccharide (LPS, positive control), phosphate-buffered saline (PBS, negative control) or colloidal gold suspension (negative particular control). After different survival times (1, 8 or 12 weeks), brain slices from the cerebral cortex or substantia nigra (SN, 1 week) were stained with Iba-1 and/or GFAP antibody to monitor microglial and astrocytic reaction, and with tyrosine hydroxylase (TH) to monitor dopaminergic cell survival (SN group only). The injection of LPS induced a strong inflammatory response in the cortex as well in the substantia nigra. Similar results could be obtained after NM injection, while the injection of PBS or gold suspension showed only moderate or no glial activation. However, the inflammatory response declined during the time course. In the SN group, there was, apart from strong microglia activation, a significant dopaminergic cell loss after 1 week of survival time. Our findings clearly indicate that extracellular NM could be one of the key molecules leading to microglial activation and neuronal cell death in the substantia nigra. This may be highly relevant to the elucidation of therapeutic strategies in PD.     

Chemokines play a critical role in the cross-regulation of Th1 and Th17 immune responses in murine crescentic glomerulonephritis

Th1 and Th17 subtype effector CD4(+) T cells are thought to play a critical role in the pathogenesis of human and experimental crescentic glomerulonephritis. The time course, mechanism, and functions of Th1 and Th17 cell recruitment, and their potential interaction in glomerulonephritis, however, remain to be elucidated. We performed interventional studies using IL-17- and IFN-γ-gene-deficient mice, as well as neutralizing antibodies that demonstrated the importance of the Th17-mediated immune response during the early phase of the disease. At a later stage, we found that Th1 cells were critical mediators of renal tissue injury. Early recruitment of IL-17-producing Th17 cells triggered expression of the chemokine CXCL9 in the kidney that drove the infiltration of Th1 cells bearing its receptor CXCR3. At a later stage, Th1 cell-derived IFN-γ was found to inhibit local chemokine CCL20 expression, acting through its receptor CCR6 on Th17 cells, thereby limiting the renal Th17 immune response. Thus, our findings provide mechanistic evidence for a cytokine-chemokine-driven feedback loop that orchestrates the observed differential Th1 and Th17 cell infiltration into the inflamed kidney. This contributes to the observed time-dependent function of these two major pathogenic effector CD4(+) T cell subsets in crescentic glomerulonephritis.