Dying neurons in thalamus of asphyxiated term newborns and rats are autophagic.

OBJECTIVE: Neonatal hypoxic-ischemic encephalopathy (HIE) still carries a high burden by its mortality and long-term neurological morbidity in survivors. Apart from hypothermia, there is no acknowledged therapy for HIE, reflecting the lack of mechanistic understanding of its pathophysiology. (Macro)autophagy, a physiological intracellular process of lysosomal degradation, has been proposed to be excessively activated in excitotoxic conditions such as HIE. The present study examines whether neuronal autophagy in the thalamus of asphyxiated human newborns or P7 rats is enhanced and related to neuronal death processes. METHODS: Neuronal autophagy and cell death were evaluated in the thalamus (frequently injured in severe HIE) of both human newborns who died after severe HIE (n = 5) and P7 hypoxic-ischemic rats (Rice-Vannuci model). Autophagic (LC3, p62), lysosomal (LAMP1, cathepsins), and cell death (TUNEL, caspase-3) markers were studied by immunohistochemistry in human and rat brain sections, and by additional methods in rats (immunoblotting, histochemistry, and electron microscopy). RESULTS: Following severe perinatal asphyxia in both humans and rats, thalamic neurons displayed up to 10-fold (p < 0.001) higher numbers of autophagosomes and lysosomes, implying an enhanced autophagic flux. The highly autophagic neurons presented strong features of apoptosis. These findings were confirmed and elucidated in more detail in rats. INTERPRETATION: These results show for the first time that autophagy is enhanced in severe HIE in dying thalamic neurons of human newborns, as in rats. Experimental neuroprotective strategies targeting autophagy could thus be a promising lead to follow for the development of future therapeutic approaches.

Role of Waddlia chondrophila placental infection in miscarriage.

Waddlia chondrophila is an intracellular bacterium suspected to cause human and bovine abortion. We confirmed an association between antibodies against W. chondrophila and human miscarriage and identified this organism in placenta or genital tract of women who had had miscarriages. These results suggest a possible role of W. chondrophila infection in miscarriage.

Intestinal spirochetosis mimicking inflammatory bowel disease in children.

BACKGROUND: Intestinal spirochetosis is an unusual infection in children and its clinical significance in humans is uncertain. The presence of these microorganisms in humans is well-known since the late 1800's and was first described in 1967 by Harland and Lee by electron microscopy. CASE PRESENTATION: This article reports the findings of one pediatric case, review of the current literature, and an overview of therapeutic options. CONCLUSION: A high degree of suspicion is required in cases presenting with abdominal pain, chronic diarrhoea and/or hematochezia associated with a normal endoscopic examination, thus emphasizing the importance of multiple biopsies throughout the colon.

Prenatal diagnosis of congenital lung malformations.

Prenatal diagnosis of congenital lung anomalies has increased in recent years as imaging methods have benefitted from technical improvements. The purpose of this pictorial essay is to illustrate typical imaging findings of a wide spectrum of congenital lung anomalies on prenatal US and MRI. Moreover, we propose an algorithm based on imaging findings to facilitate the differential diagnosis, and suggest a follow-up algorithm during pregnancy and in the immediate postnatal period.

Role of Chlamydia trachomatis in miscarriage.

To determine the role of Chlamydia trachomatis in miscarriage, we prospectively collected serum, cervicovaginal swab specimens, and placental samples from 386 women with and without miscarriage. Prevalence of immunoglobulin G against C. trachomatis was higher in the miscarriage group than in the control group (15.2% vs. 7.3%; p = 0.018). Association between C. trachomatis-positive serologic results and miscarriage remained significant after adjustment for age, origin, education, and number of sex partners (odds ratio 2.3, 95% confidence interval 1.1-4.9). C. trachomatis DNA was more frequently amplified from products of conception or placenta from women who had a miscarriage (4%) than from controls (0.7%; p = 0.026). Immunohistochemical analysis confirmed C. trachomatis in placenta from 5 of 7 patients with positive PCR results, whereas results of immunohistochemical analysis were negative in placenta samples from all 8 negative controls tested. Associations between miscarriage and serologic/molecular evidence of C. trachomatis infection support its role in miscarriage.

16q24.1 microdeletion in a premature newborn: usefulness of array-based comparative genomic hybridization in persistent pulmonary hypertension of the newborn.

OBJECTIVE: Report of a 16q24.1 deletion in a premature newborn, demonstrating the usefulness of array-based comparative genomic hybridization in persistent pulmonary hypertension of the newborn and multiple congenital malformations. DESIGN: Descriptive case report. SETTING: Genetic department and neonatal intensive care unit of a tertiary care children's hospital. INTERVENTIONS: None. PATIENT: We report the case of a preterm male infant, born at 26 wks of gestation. A cardiac malformation and bilateral hydronephrosis were diagnosed at 19 wks of gestation. Karyotype analysis was normal, and a 22q11.2 microdeletion was excluded by fluorescence in situ hybridization analysis. A cesarean section was performed due to fetal distress. The patient developed persistent pulmonary hypertension unresponsive to mechanical ventilation and nitric oxide treatment and expired at 16 hrs of life. MEASUREMENTS AND MAIN RESULTS: An autopsy revealed partial atrioventricular canal malformation and showed bilateral dilation of the renal pelvocaliceal system with bilateral ureteral stenosis and annular pancreas. Array-based comparative genomic hybridization analysis (Agilent oligoNT 44K, Agilent Technologies, Santa Clara, CA) showed an interstitial microdeletion encompassing the forkhead box gene cluster in 16q24.1. Review of the pulmonary microscopic examination showed the characteristic features of alveolar capillary dysplasia with misalignment of pulmonary veins. Some features were less prominent due to the gestational age. CONCLUSIONS: Our review of the literature shows that alveolar capillary dysplasia with misalignment of pulmonary veins is rare but probably underreported. Prematurity is not a usual presentation, and histologic features are difficult to interpret. In our case, array-based comparative genomic hybridization revealed a 16q24.1 deletion, leading to the final diagnosis of alveolar capillary dysplasia with misalignment of pulmonary veins. It emphasizes the usefulness of array-based comparative genomic hybridization analysis as a diagnostic tool with implications for both prognosis and management decisions in newborns with refractory persistent pulmonary hypertension and multiple congenital malformations.

Isolation and in vitro chondrogenic potential of human foetal spine cells.

Cell therapy for nucleus pulposus (NP) regeneration is an attractive treatment for early disc degeneration as shown by studies using autologous NP cells or stem cells. Another potential source of cells is foetal cells. We investigated the feasibility of isolating foetal cells from human foetal spine tissues and assessed their chondrogenic potential in alginate bead cultures. Histology and immunohistochemistry of foetal tissues showed that the structure and the matrix composition (aggrecan, type I and II collagen) of foetal intervertebral disc (IVD) were similar to adult IVD. Isolated foetal cells were cultured in monolayer in basic media supplemented with 10% Fetal Bovine Serum (FBS) and from each foetal tissue donation, a cell bank of foetal spine cells at passage 2 was established and was composed of around 2000 vials of 5 million cells. Gene expression and immunohistochemistry of foetal spine cells cultured in alginate beads during 28 days showed that cells were able to produce aggrecan and type II collagen and very low level of type I and type X collagen, indicating chondrogenic differentiation. However variability in matrix synthesis was observed between donors. In conclusion, foetal cells could be isolated from human foetal spine tissues and since these cells showed chondrogenic potential, they could be a potential cell source for IVD regeneration.

Familial occurrence of an association of multiple intestinal atresia and choanal atresia: a new syndrome?

We report on two familial cases from a non-consanguineous marriage, presenting multiple intestinal and choanal atresia. Massive hydramnios and dilatation of the bowel were observed at 29 weeks of gestation during routine ultrasound scan of a healthy mother. The fetal karyotype was normal and cystic fibrosis screening was negative. Regular scans were performed throughout the pregnancy. The child was born at 34 weeks gestation. Choanal atresia was diagnosed at birth and abdominal investigations showed multiple atresia interesting both the small bowel and the colon. Further interventions were necessary because of recurrent obstructions. During the following pregnancy, a dilatation of the fetal intestinal tract was detected by ultrasonography at 27 weeks of gestation. Pregnancy was interrupted. Post-mortem examination of the fetus confirmed the stenosis of long segments of the small intestine associated with areas of colonic atresia. In both cases, histology and distribution were consistent with those reported in hereditary multiple intestinal atresia (HMIA). An association between multiple intestinal and choanal atresia has never been reported. We suggest it could correspond to a new autosomal recessive entity for which cytogenetic investigations and high-resolution array CGH revealed no visible anomalies.

Molecular risk stratification of medulloblastoma patients based on immunohistochemical analysis of MYC, LDHB, and CCNB1 expression.

PURPOSE: Medulloblastoma is the most common malignant embryonal brain tumor in children. The current clinical risk stratification to select treatment modalities is not optimal because it does not identify the standard-risk patients with resistant disease or the unknown number of high-risk patients who might be overtreated with current protocols. The aim of this study is to improve the risk stratification of medulloblastoma patients by using the expression of multiple prognostic markers in combination with current clinical parameters. EXPERIMENTAL DESIGN: Candidate prognostic markers were selected from literature or from medulloblastoma expression data. Selected genes were immunohistochemically analyzed for their prognostic value using medulloblastoma tissue arrays containing 124 well-characterized patient samples. RESULTS: Protein expression analyses showed that the combined expression of three genes was able to predict survival in medulloblastoma patients. Low MYC expression identified medulloblastoma patients with a very good outcome. In contrast, concomitant expression of LDHB and CCNB1 characterized patients with a very poor outcome. Multivariate analyses showed that both expression of MYC and the LDHB/CCNB1 gene signature were strong prognostic markers independent of the clinical parameters metastasis and residual disease. Combined analysis of clinical and molecular markers enabled greater resolution of disease risk than clinical factors alone. CONCLUSIONS: A molecular risk stratification model for medulloblastoma patients is proposed based on the signature of MYC, LDHB, and CCNB1 expression. Combined with clinical variables, the model may provide a more accurate basis for targeting therapy in children with this disease.

Hirschsprung's disease: the "Swiss roll" technique revisited.

PURPOSE: During pull-through for Hirschsprung's disease (HSCR), the assessment of innervation is mainly based on the presence of ganglion cells when conventional Hematoxylin and Eosin (HE) staining is used. In hypoganglionosis, the evaluation is difficult. We adapted a standardized methodology for the examination of resected bowel after HSCR surgery, using the technique described by Moolenbeek on rodent intestine and later by Meier-Ruge in children. We have analysed the entire innervation of surgically resected bowels and compared the results with the follow up of patients. METHODS: Three longitudinal strips of colon were harvested from the mesenteric, anti-mesenteric and intermediate part in the whole length of resected colon of six patients with HSCR. Each strip was divided into two parts. One of the contiguous strips was assessed with HE and Hematoxylin-Phloxin-Safran, and the other one with acetylcholinesterase (AChE) histochemistry. We analyzed the distribution of ganglion cells and nerve arrangement along the strips with both techniques and compared the results obtained in the three different regions of the bowel. RESULTS: There was no significant difference in the pattern of innervation circumferentially. There was a correlation between a progressive increase of AChE activity and nerve hypertrophy and a decrease of ganglion cells from the proximal to the distal part of the resected colon in the submucosa and the myenteric plexus. Nerve hypertrophy and AChE-positive reaction in the mucosa were found at the resection border in patients who presented postoperative complications. CONCLUSIONS: Simultaneous assessment of nerve cells, nerve fibers and AChE activity is important in the evaluation of the innervation of the bowel segment proximal to the aganglionic zone. The method described is feasible and can be adapted to older children and adults with larger bowels. These results point out the importance of assessing nerve fibers in intraoperative biopsies during pull-through procedures to prevent uncomplete surgical bowel resection.

Placental immaturity, endocardial fibroelastosis and fetal hypoxia.

We describe a term newborn who, after a normal gestational course, presented at birth with absent cardiac activity and no spontaneous breathing. Death occurred within 30 h. Autopsy revealed placental villous immaturity, multiple acute hypoxic lesions, but also chronic hypoxic lesions like endocardial fibroelastosis. This striking association of endocardial fibroelastosis and placental villous immaturity is reviewed and correlated with 2 other cases of placental villous immaturity that led to in utero death at 39 and 41 weeks of gestation. Placental villous immaturity must be suspected and looked for by both pediatricians and obstetricians in every case of stillbirth or perinatal asphyxia of unclear origin. In order to minimize the risk of recurrence in further pregnancies, elective cesarean section may be considered.

Role of DNA content analysis and immunohistochemistry in the evaluation of the risk of unfavourable outcome in Wilms' tumours.

BACKGROUND: Wilms' tumour (WT) is the most common solid tumour affecting young children. Its histological diversity leads to difficulties in predicting the outcome. MATERIALS AND METHODS: Image analysis cytometry and immunohistochemistry with a selected panel of antibodies were performed in 23 cases of WT considered of intermediate risk according to the revised International Society of Pediatric Oncology (SIOP) working classification of renal tumours of childhood. In this series, a tumour was considered aggressive according to its propensity for metastases or its recurrence. RESULTS: Out of the 14 non-aggressive WT, 4 were found to be diploid and 10 were aneuploid including 6 that were heterogeneous for DNA-ploidy. All the tumours presented a low proliferative index and were negative for p53 and p57(kip2) immunostaining. Out of the 9 aggressive tumours, all were aneuploid and 4 were found to be heterogeneous for DNA-ploidy. They all presented a high degree of cell proliferation and 7 were positive for p53 immunostaining. Only two were positive for the p57(kip2) marker. The only fatal case revealed an aneuploid-homogeneous DNA-ploidy analysis, was p53 and p57(kip2) positive and presented a high cell proliferation index. CONCLUSION: A significant correlation between the presence of focal DNA-aneuploidy in Wilms' tumours and adverse prognosis is not established, but some immunohistochemical markers may be useful for the clinical evaluation of these tumours and to help in predicting the risk of an unfavourable outcome.

Significance of hemorrhagic endovasculitis in placentae from stillbirths.

Hemorrhagic endovasculitis (HEV) is an alteration of fetal chorionic blood vessels within the placenta. This disruption of the integrity of the parietal blood vessels has been reported in association with poor pregnancy outcomes and perinatal complications. In stillbirths, its presence has been implicated as part of the mechanism of the cessation of fetal circulation in postmortem retention of the placenta. The purpose of this study was to give an interpretation of this type of blood vessel damage: whether it is related to the arrest of blood circulation after stillbirth, or whether it constitutes an alteration of the integrity of placental chorionic circulation contributing to the death of the fetus. From 1232 reviewed placentae, we analyzed 46 cases with HEV, 70% from livebirths and 30% from stillbirths. The most frequent lesions encountered in placentae were categorized microscopically. The distribution of these lesions in placentae from livebirth and stillbirth pregnancies was then described. In association with HEV, villitis of unknown etiology and chorionic vessel thrombi occurred more frequently in placentae from livebirths. Fragmented erythrocytes and erythroblasts were more frequent in placentae from stillbirths. Our study demonstrates that HEV is more consistently associated with other placental lesions in liveborn than in stillborn infants.

[Pediatric pathology: a child is not an adult in miniature form]. / La pathotogie pédiatrique: un enfant n'est pas un adulte miniature.

In pediatric pathology, diagnosis explores major aspects of childhood disorders ranging from malformative syndromes to neoplasm's and forensic pathology. For this mental gymnastics, knowledge of the different morphological and physiological process of development and adaptation are essential. Furthermore, new adjuvant technology ensure a more precise diagnosis by the pediatric pathologist. A review of some particular situations in child are illustrated below.

Image cytometry: an aid for cytological diagnosis of pleural effusions.

The conventional cytology rate for identification of neoplastic cells in effusions is about 60%. The rate of diagnostically equivocal effusions in routine cytology is dependent on the volume of effusion examined, type of preparation and staining, experience of the examiner, and application of ancillary methods. The aim of our study was to confirm the role of image cytometry analysis (DNA-ploidy) on pleural effusions. In this retrospective study based on 42 available cases with a histological diagnosis, we have examined 13 reactive mesothelial proliferations and 29 cases of malignant tumors (adenocarcinoma [ACA] or mesothelioma). The smears collected were submitted to the image analysis following a-three step protocol (smears stained with the Papanicolaou method were destained and then restained with Feulgen staining and finally analysed using image analysis cytometry). The results have shown that nonmalignant cases (reactive mesothelial proliferation) were all diploid and in contrast all aneuploid cases corresponded to malignant tumors. Only three mesotheliomas showed a diploid profile. In conclusion, these results confirm data from literature and indicate that cytometric analysis of nuclear content is a useful marker for identification of malignant cells in equivocal effusions and can be used to increase the cytological sensitivity in doubtful mesothelial proliferations.

Beta-catenin expression and its association with prognostic factors in adenocarcinoma developed in Barrett esophagus.

The majority of the adenocarcinomas arising in Barrett esophagus manifest clinically at an advanced stage and have a poor prognosis. As a result of this poor prognosis, much attention has been directed toward the exploration of markers for neoplastic progression in Barrett esophagus. The objective of the present study was to determine the expression of beta-catenin by immunohistochemical analysis in 70 adenocarcinomas developed in Barrett esophagus and to examine its relationship to various prognostic factors currently in use. Abnormal beta-catenin expression, consisting of the loss of membranous staining and the appearance of the nuclear staining, was found in 43 cases (61%). Of patients with the 43 tumors showing abnormal beta-catenin expression, 25 (58%) survived more than 1 year. In contrast, only 7 (26%) of 27 patients with tumors showing normal beta-catenin expression survived longer than 1 year. Most of the superficial (Tis-T1) tumors (83% [10/12]) exhibited abnormal beta-catenin expression compared with only 53% (31/58) in the T2-T3 group. These results suggest a possible correlation among beta-catenin expression, tumor stage, and length of survival as prognostic factors in patients with adenocarcinoma in Barrett esophagus.

DNA-ploidy in advanced gastric carcinoma is less heterogeneous than in early gastric cancer.

This analysis of DNA-ploidy heterogeneity in advanced gastric carcinomas is consistent with the hypothesis of the emergence of a single aneuploid cell clone as a crucial mechanism in the progression from early gastric carcinoma to advanced gastric cancer. The prognostic value of DNA-ploidy in gastric cancers has been a matter of controversy. Tumour DNA-ploidy heterogeneity, the presence within the same tumour of multiple stemlines differing in DNA content, has been described in various tumours including gastric cancers. The occurrence of such heterogeneity has been accepted as an explanation for the divergent DNA-ploidy results in this type of tumours. A previous study of early gastric cancers suggested that in pure diploid superficial carcinomas, genetic instability might lead to a cell clone which has undergone a ploidy shift and is more aggressive. If so, this would initially result in DNA-ploidy heterogeneity. Proliferative dominance of the aneuploid clone could eventually evolve to a homogeneous aneuploid tumour. In order to test this hypothesis, we studied DNA-aneuploidy and DNA-ploidy heterogeneity in advanced gastric carcinomas. We performed DNA cytophotometry on multiple samples collected from 16 advanced gastric carcinomas and found 15 DNA-aneuploid tumours (94%) and one diploid tumour. Multiple DNA-stemlines were found in 4 cases (26%). Analysis of proliferative activity performed on the same samples revealed higher proliferation rate in DNA-ploidy homogeneous tumours than in aneuploid heterogeneous tumours. Heterogeneous tumours did not overexpress p53. These results confirm that DNA-aneuploidy is frequent in advanced gastric cancer and demonstrate that a majority of these aneuploid tumours are not DNA-ploidy heterogeneous. Furthermore, the higher proliferative activity in homogeneous-aneuploid carcinomas and their more frequent overexpression of p53 support the hypothesis that in gastric cancer tumour progression implies the development of a dominant and more aggressive (higher proliferative activity, p53 overexpression) aneuploid cell clone.

The channel-activating protease CAP1/Prss8 is required for placental labyrinth maturation.

The serine protease CAP1/Prss8 is crucial for skin barrier function, lung alveolar fluid clearance and has been unveiled as diagnostic marker for specific cancer types. Here, we show that a constitutive knockout of CAP1/Prss8 leads to embryonic lethality. These embryos presented no specific defects, but it is during this period, and in particular at E13.5, that wildtype placentas show an increased expression of CAP1/Prss8, thus suggesting a placental defect in the knockout situation. The placentas of knockout embryos exhibited significantly reduced vascular development and incomplete cellular maturation. In contrary, epiblast-specific deletion of CAP1/Prss8 allowed development until birth. These CAP1/Prss8-deficient newborns presented abnormal epidermis, and died soon after birth due to impaired skin function. We thus conclude that a late placental insufficiency might be the primary cause of embryonic lethality in CAP1/Prss8 knockouts. This study highlights a novel and crucial role for CAP1/Prss8 in placental development and function.