The RNA-binding protein RBPMS2 regulates development of gastrointestinal smooth muscle.

BACKGROUND & AIMS: Gastrointestinal development requires regulated differentiation of visceral smooth muscle cells (SMCs) and their contractile activities; alterations in these processes might lead to gastrointestinal neuromuscular disorders. Gastrointestinal SMC development and remodeling involves post-transcriptional modification of messenger RNA. We investigated the function of the RNA-binding protein for multiple splicing 2 (RBPMS2) during normal development of visceral smooth muscle in chicken and expression of its transcript in human pathophysiological conditions. METHODS: We used avian replication-competent retroviral misexpression approaches to analyze the function of RBPMS2 in vivo and in primary cultures of chicken SMCs. We analyzed levels of RBPMS2 transcripts in colon samples from pediatric patients with Hirschsprung's disease and patients with chronic pseudo obstruction syndrome (CIPO) with megacystis. RESULTS: RBPMS2 was expressed strongly during the early stage of visceral SMC development and quickly down-regulated in differentiated and mature SMCs. Misexpression of RBPMS2 in differentiated visceral SMCs induced their dedifferentiation and reduced their contractility by up-regulating expression of Noggin, which reduced activity of bone morphogenetic protein. Visceral smooth muscles from pediatric patients with CIPO expressed high levels of RBPMS2 transcripts, compared with smooth muscle from patients without this disorder. CONCLUSIONS: Expression of RBPMS2 is present in visceral SMC precursors. Sustained expression of RBPMS2 inhibits the expression of markers of SMC differentiation by inhibiting bone morphogenetic protein activity, and stimulates SMC proliferation. RBPMS2 transcripts are up-regulated in patients with CIPO; alterations in RBPMS2 function might be involved in digestive motility disorders, particularly those characterized by the presence of muscular lesions (visceral myopathies).

High expression of the RNA-binding protein RBPMS2 in gastrointestinal stromal tumors.

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal neoplasms of the gastrointestinal tract and are often associated with KIT or PDGFRA gene mutations. GIST cells might arise from the interstitial cells of Cajal (ICCs) or from a mesenchymal precursor that is common to ICCs and smooth muscle cells (SMCs). Here, we analyzed the mRNA and protein expression of RNA-Binding Protein with Multiple Splicing-2 (RBPMS2), an early marker of gastrointestinal SMC precursors, in human GISTs (n=23) by in situ hybridization, quantitative RT-PCR analysis and immunohistochemistry. The mean RBPMS2 mRNA level in GISTs was 42-fold higher than in control gastrointestinal samples (p<0.001). RBPMS2 expression was not correlated with KIT and PDGFRA expression levels, but was higher in GISTs harboring KIT mutations than in tumors with wild type KIT and PDGFRA or in GISTs with PDGFRA mutations that were characterized by the lowest RBPMS2 levels. Moreover, RBPMS2 levels were 64-fold higher in GIST samples with high risk of aggressive behavior than in adult control gastrointestinal samples and 6.2-fold higher in high risk than in low risk GIST specimens. RBPMS2 protein level was high in 87% of the studied GISTs independently of their histological classification. Finally, by inhibiting the KIT signaling pathway in GIST882 cells, we show that RBPMS2 expression is independent of KIT activation. In conclusion, RBPMS2 is up-regulated in GISTs compared to normal adult gastrointestinal tissues, indicating that RBPMS2 might represent a new diagnostic marker for GISTs and a potential target for cancer therapy.

Cobblestone lissencephaly: neuropathological subtypes and correlations with genes of dystroglycanopathies.

Cobblestone lissencephaly represents a peculiar brain malformation with characteristic radiological anomalies, defined as cortical dysplasia combined with dysmyelination, dysplastic cerebellum with cysts and brainstem hypoplasia. Cortical dysplasia results from neuroglial overmigration into the arachnoid space, forming an extracortical layer, responsible for agyria and/or 'cobblestone' brain surface and ventricular enlargement. The underlying mechanism is a disruption of the glia limitans, the outermost layer of the brain. Cobblestone lissencephaly is pathognomonic of a continuum of autosomal recessive diseases with cerebral, ocular and muscular deficits, Walker-Warburg syndrome, muscle-eye-brain and Fukuyama muscular dystrophy. Mutations in POMT1, POMT2, POMGNT1, LARGE, FKTN and FKRP genes attributed these diseases to α-dystroglycanopathies. However, studies have not been able to identify causal mutations in the majority of patients and to establish a clear phenotype/genotype correlation. Therefore, we decided to perform a detailed neuropathological survey and molecular screenings in 65 foetal cases selected on the basis of histopathological criteria. After sequencing the six genes of α-dystroglycanopathies, a causal mutation was observed in 66% of cases. On the basis of a ratio of severity, three subtypes clearly emerged. The most severe, which we called cobblestone lissencephaly A, was linked to mutations in POMT1 (34%), POMT2 (8%) and FKRP (1.5%). The least severe, cobblestone lissencephaly C, was linked to POMGNT1 mutations (18%). An intermediary type, cobblestone lissencephaly B, was linked to LARGE mutations (4.5%) identified for the first time in foetuses. We conclude that cobblestone lissencephaly encompasses three distinct subtypes of cortical malformations with different degrees of neuroglial ectopia into the arachnoid space and cortical plate disorganization regardless of gestational age. In the cerebellum, histopathological changes support the novel hypothesis that abnormal lamination arises from a deficiency in granule cells. Our studies demonstrate the positive impact of histoneuropathology on the identification of α-dystroglycanopathies found in 66% of cases, while with neuroimaging criteria and biological values, mutations are found in 32-50% of patients. Interestingly, our morphological classification was central in the orientation of genetic screening of POMT1, POMT2, POMGNT1, LARGE and FKRP. Despite intensive research, one-third of our cases remained unexplained; suggesting that other genes and/or pathways may be involved. This material offers a rich resource for studies on the affected neurodevelopmental processes of cobblestone lissencephaly and on the identification of other responsible gene(s)/pathway(s).

Antenatal spectrum of CHARGE syndrome in 40 fetuses with CHD7 mutations.

BACKGROUND: CHARGE syndrome is a rare, usually sporadic disorder of multiple congenital anomalies ascribed to a CHD7 gene mutation in 60% of cases. Although the syndrome is well characterised in children, only one series of 10 fetuses with CHARGE syndrome has been reported to date. Therefore, we performed a detailed clinicopathological survey in our series of fetuses with CHD7 mutations, now extended to 40 cases. CHARGE syndrome is increasingly diagnosed antenatally, but remains challenging in many instances. METHOD: Here we report a retrospective study of 40 cases of CHARGE syndrome with a CHD7 mutation, including 10 previously reported fetuses, in which fetal or neonatal clinical, radiological and histopathological examinations were performed. RESULTS: Conversely to postnatal studies, the proportion of males is high in our series (male to female ratio 2.6:1) suggesting a greater severity in males. Features almost constant in fetuses were external ear anomalies, arhinencephaly and semicircular canal agenesis, while intrauterine growth retardation was never observed. Finally, except for one, all other mutations identified in our antenatal series were truncating, suggesting a possible phenotype-genotype correlation. CONCLUSIONS: Clinical analysis allowed us to refine the clinical description of CHARGE syndrome in fetuses, describe some novel features and set up diagnostic criteria in order to help the diagnosis of CHARGE syndrome after termination of pregnancies following the detection of severe malformations.

Immunohistochemical analysis of bone morphological protein signaling pathway in human myometrium.

We assessed by immunohistochemistry the expression of the phosphorylated (activated) form of Smad1 and 5 (P-SMAD1/5), of Noggin and of two smooth muscle cell markers (α-SMA and SM22) in a series of human myometrium samples and in a smooth muscle cell line derived from human myometrium (HUt-SMC, PromoCell, USA). Myometrium samples were removed from two cadavers (a fetus at 26 weeks of gestation and a neonate) and from ten non-menopausal women who underwent hysterectomy for adenomyosis and leiomyoma. P-SMAD1/5 expression was never detected in myometrium (both normal and pathological specimens), but only as a nuclear positive staining in glandular and luminal epithelial cells in sections in which also the endometrial mucosa was present. Noggin was strongly expressed especially in myometrium and adenomyosis samples from non-menopausal patients in comparison to the neonatal and fetal myometrium specimens in which muscle cells were less positive. In more than 95% of HUt-SMCs, α-SMA and Desmin were co-expressed, indicating a pure smooth muscle phenotype. When progesterone was added to the culture medium, no P-SMAD1/5 expression was detected, whereas the expression Noggin and SM22, a marker of differentiated smooth muscle cells, increased by 3 fold (p=0.002) and 4.3 fold (p=0.001), respectively (p=0.002). Our results suggest that, in non-menopausal normal human myometrium, the BMP pathway might be inhibited and that this inhibition might be enhanced by progesterone, which increases the differentiation of smooth muscle cells (SM22 levels). These findings could help in the identification of new mechanisms that regulate uterine motility.

Posterior fossa solitary fibrous tumour: report of a fetal case and review of the literature.

We report a case of solitary fibrous tumour (SFT) involving the posterior fossa in a fetus of 25 weeks' gestation. SFT is a rare mesenchymal neoplasm, arising in various locations including the meninges. After disclosure of severe ventriculomegaly and posterior fossa mass measuring 45 mm in diameter, termination of pregnancy was performed in accordance with French legislation. Our neuropathological study revealed a tumour covered by meninges, with severe compression of the cerebellum and the brain stem. Microscopically, the tumour was highly cellular, made of packed small fusiform cells with branching vasculature and consistent expression of CD34. No extraneurological lesion was noted. Except cysts and vascular malformations, posterior fossa tumours have been exceptionally reported in fetuses. SFT was distinguished from hemangiopericytoma. In spite of the fact these tumours share many similarities, some criteria such as the staining pattern for CD34 instead indicated a SFT. Histology was distinctive of hemangioblastoma and primitive neuroectodermal tumour. The prognosis of solitary fibrous tumour, which is usually a benign tumour, was there worsened by the precocity of the onset and the local invasion causing disruption of the cerebellum, compression of the brain stem and severe ventriculomegaly.

New concept for immediate breast reconstruction for invasive cancers: feasibility, oncological safety and esthetic outcome of post-neoadjuvant therapy immediate breast reconstruction versus delayed breast reconstruction: a prospective pilot study.

Feasibility and oncological safety of post-adjuvant skin-sparing mastectomy (SSM) plus immediate breast reconstruction (IBR) cannot be evaluated by randomized trials. However, comparative study could modify guidelines for the oncosurgical treatment of invasive breast cancer. Our study compared the feasibility, oncological safety and esthetic outcome of SSM plus latissimus dorsi (LD) flap IBR after chemotherapy (CT) and radiotherapy (RT) with the standard management for invasive breast cancer: mastectomy as primary treatment, adjuvant CT and RT, and LD flap delayed breast reconstruction (DBR). Twenty-six selected patients with stages IIA-IIIA breast cancer were offered post-neoadjuvant SSM plus IBR with LD flap plus implant (IBR group). Seventy-eight other patients had primary mastectomy, adjuvant CT and RT, and LD-assisted DBR (DBR group). After 4.1 years (range 1-8) of follow-up, feasibility, oncological safety, and esthetic outcome were compared. Sixteen (61%) early complications were reported for the IBR group versus 44 (56%) for the DBR group (P = 0.645). Early implant loss was 0% in IBR versus 12% in DBR. IBR had 8 (30%) late complications versus 17 (21%) for DBR (P = 0.362). Capsular contracture and reconstruction failure rates were similar. Local recurrence was 7.7% (2/26) in IBR and 6.4% (5/78) in DBR (P = 0.823). Cosmetic evaluation by independent physicians and by the patients themselves was identical in the two groups. Our concept provides a basis for offering more women the opportunity to elect for immediate reconstruction, even in the setting of radiation therapy.

Array-CGH analysis indicates a high prevalence of genomic rearrangements in holoprosencephaly: an updated map of candidate loci.

Holoprosencephaly (HPE) is the most frequent malformation of the brain. To date, 12 different HPE loci and 8 HPE genes have been identified from recurrent chromosomal rearrangements or from the sequencing of genes from Nodal and SHH pathways. Our cohort of HPE patients presents a high genetic heterogeneity. Point mutations were found in SHH, ZIC2, SIX3, and TGIF genes in about 20% of cases (with 10% in SHH). Deletions in these same genes were found in 7.5% of the patients and 4.4% presented with other subtelomeric gain or losses. Consequently, the molecular basis of HPE remains unknown in 70% of our cohorts. To detect new HPE candidate genes, we used array-CGH to refine the previous karyotype based HPE loci map. We analyzed 111 HPE patients with high-performance Agilent oligonucleotidic arrays and found that 28 presented anomalies involving known or new potential HPE loci located on different chromosomes but with poor redundancy. This study showed an impressive rate of 19 patients among 111 with de novo chromosomal anomalies giving evidence that microrearrangements could be a major molecular mechanism in HPE. Additionally, this study opens new insights on HPE candidate genes identification giving an updated HPE candidate loci map.

A human spinal cord cell promotes motoneuron survival and maturation in vitro.

Primary cultures of motoneurons represent a good experimental model for studying mechanisms underlying certain spinal cord pathologies, such as amyotrophic lateral sclerosis and spinal bulbar muscular atrophy (Kennedy's disease). However, a major problem with such culture systems is the relatively short cell survival times, which limits the extent of motoneuronal maturation. In spite of supplementing culture media with various growth factors, it remains difficult to maintain motoneurons viable longer than 10 days in vitro. This study employs a new approach, in which rat motoneurons are plated on a layer of cultured cells derived from newborn human spinal cord. For all culture periods, more motoneurons remain viable in such cocultures compared with control monocultures. Moreover, although no motoneurons survive in control cultures after 22 days, viable motoneurons were observed in cocultures even after 7 weeks. Although no significant difference in neurite length was observed between 8-day mono- and cocultures, after 22 and 50 days in coculture motoneurons had a very mature morphology. They extended extremely robust, very long neurites, which formed impressive branched networks. Data obtained using a system in which the spinal cord cultures were separated from motoneurons by a porous polycarbonate filter suggest that soluble factors released from the supporting cells are in part responsible for the beneficial effects on motoneurons. Several approaches, including immunocytochemistry, immunoblotting, and electron microscopy, indicated that these supporting cells, capable of extending motoneuron survival and enhancing neurite growth, had an undifferentiated or poorly differentiated, possibly mesenchymal phenotype.

First report of a necrotising fasciitis of the breast following a core needle biopsy.

Necrotising fasciitis (NF) is the most aggressive form of soft tissue infection. We report the first case of NF of the breast following a core needle biopsy. Aggressive management including surgical debridement and vacuum therapy allowed wound healing and breast conservation.

Human PDCD2L Is an Export Substrate of CRM1 That Associates with 40S Ribosomal Subunit Precursors.

Protein arginine methyltransferase 3 (PRMT3) forms a stable complex with 40S ribosomal protein S2 (RPS2) and contributes to ribosome biogenesis. However, the molecular mechanism by which PRMT3 influences ribosome biogenesis and/or function still remains unclear. Using quantitative proteomics, we identified human programmed cell death 2-like (PDCD2L) as a novel PRMT3-associated protein. Our data suggest that RPS2 promotes the formation of a conserved extraribosomal complex with PRMT3 and PDCD2L. We also show that PDCD2L associates with 40S subunit precursors that contain a 3'-extended form of the 18S rRNA (18S-E pre-rRNA) and several pre-40S maturation factors. PDCD2L shuttles between the nucleus and the cytoplasm in a CRM1-dependent manner using a leucine-rich nuclear export signal that is sufficient to direct the export of a reporter protein. Although PDCD2L is not required for the biogenesis and export of 40S ribosomal subunits, we found that PDCD2L-null cells accumulate free 60S ribosomal subunits, which is indicative of a deficiency in 40S subunit availability. Our data also indicate that PDCD2L and its paralog, PDCD2, function redundantly in 40S ribosomal subunit production. Our findings uncover the existence of an extraribosomal complex consisting of PDCD2L, RPS2, and PRMT3 and support a role for PDCD2L in the late maturation of 40S ribosomal subunits.

Correlation between placental bacterial culture results and histological chorioamnionitis: a prospective study on 376 placentas.

OBJECTIVE: To study the correlation between the bacteriological and histopathological findings in placentas from women with suspected or proven chorioamnionitis (CA). METHODS: Over a 1-year period, 376 placentas were prospectively collected and processed for bacteriological and pathological studies in cases of confirmed or suspected maternal or neonatal infection. RESULTS: Histological CA was diagnosed in 26.9% of placentas (101/376), and 27.7% (28/101) of these placentas had positive bacteriological cultures. A monomicrobial culture, mainly represented by Gram-positive cocci and Gram-negative bacilli, was identified in 27% of the positive bacterial cultures. The proportion of positive cultures was higher (p=0.03) when CA was associated with funisitis, as compared with placental samples with early CA. In placentas without histological CA, bacteriological cultures were mostly negative (230/275), although pathogenic bacteria were identified in 16.3% of them (45/275). CONCLUSIONS: The histological and bacteriological results were concordant in about 70% of the examined placentas, with 61.1% negative cases (CA absent and negative bacterial cultures), and only 7.4% placentas with positive histological and bacteriological results. Discordant results (positive histology with negative bacteriology) were obtained in placentas with early CA documented by histology although possibly in relation with antibiotic prophylaxis and the presence of fastidious bacteria. Conversely, negative histology with positive bacteriology could be explained by the presence of an early-stage bacterial infection that has not yet led to detectable microscopic lesions.

Organ volume measurements: comparison between MRI and autopsy findings in infants following sudden unexpected death.

OBJECTIVE: To assess the accuracy of a semiautomated 3D volume reconstruction method for organ volume measurement by postmortem MRI. METHODS: This prospective study was approved by the institutional review board and the infants' parents gave their consent. Postmortem MRI was performed in 16 infants (1 month to 1 year of age) at 1.5 T within 48 h of their sudden death. Virtual organ volumes were estimated using the Myrian software. Real volumes were recorded at autopsy by water displacement. The agreement between virtual and real volumes was quantified following the Bland and Altman's method. RESULTS: There was a good agreement between virtual and real volumes for brain (mean difference: -0.03% (-13.6 to +7.1)), liver (+8.3% (-9.6 to +26.2)) and lungs (+5.5% (-26.6 to +37.6)). For kidneys, spleen and thymus, the MRI/autopsy volume ratio was close to 1 (kidney: 0.87±0.1; spleen: 0.99±0.17; thymus: 0.94±0.25), but with a less good agreement. For heart, the MRI/real volume ratio was 1.29±0.76, possibly due to the presence of residual blood within the heart. The virtual volumes of adrenal glands were significantly underestimated (p=0.04), possibly due to their very small size during the first year of life. The percentage of interobserver and intraobserver variation was lower or equal to 10%, but for thymus (15.9% and 12.6%, respectively) and adrenal glands (69% and 25.9%). CONCLUSIONS: Virtual volumetry may provide significant information concerning the macroscopic features of the main organs and help pathologists in sampling organs that are more likely to yield histological findings.

Prevalence and timing of pregnancy termination for brain malformations.

OBJECTIVE: To determine the prevalence and the timing of pregnancy termination relative to the type of central nervous system (CNS) malformations. Design Retrospective cohort study. SETTING: Multidisciplinary centre for prenatal diagnosis in the Languedoc-Roussillon region, France. POPULATION: A cohort of 481 pregnancy terminations performed between 2005 and 2009. METHODS: Detailed post-termination fetal and neuropathological analyses were carried out to identify the CNS malformations. Then, the prevalence and timing of pregnancy termination were assessed relative to the identified malformations. RESULTS: About one-third of pregnancy terminations (143/481) were performed for severe CNS malformations. Up to 24 weeks of gestation (WG), pregnancy terminations (56.6%) were carried out mainly for defects occurring during the two major first steps of CNS development (neurulation and differentiation of cerebral vesicles). After 24 WG, pregnancy terminations (43.3%) were mainly performed for corpus callosum agenesis (16/17), vermian agenesis (10/12) and gyral anomalies (13/15). For hindbrain malformations and gyral anomalies, there was a significant relationship between the timing of pregnancy termination and the presence of a severe ventriculomegaly at prenatal diagnosis (p=0.002 and p=0.02, respectively). CONCLUSION: By classifying CNS malformations according to the neuropathological analysis, the authors show that the timing and prevalence of pregnancy termination are distributed in a manner that is consistent with what is currently known on the development of brain. They are also influenced by the French prenatal screening policy and the variable expressivity of the brain malformations and associated lesions.

Pyruvate modifies glycolytic and oxidative metabolism of rat embryonic spinal cord astrocyte cell lines and prevents their spontaneous transformation.

This study aimed to provide detailed data on mitochondrial respiration of normal astrocyte cell lines derived from rat embryonic spinal cord. Astrocytes in early passages (EP), cultured without pyruvate for more than 35 passages, defined here as late passages (LP), undergo spontaneous transformation. To study initial steps in cell transformation, EP data were compared with those of LP cells. LP cells had reduced glycolysis, fewer mitochondria and extremely low oxidative rates, resulting from a dysfunction of complexes I and II + III of the respiratory chain. Treatment of EP cells with pyruvate until they were, by definition, LP cultures prevented transformation of these cells. Pyruvate-treated EP cells had more mitochondria than normal cells but slightly lower respiratory rates. The increase of mitochondrial content thus appears to act as a compensatory effect to maintain oxidative phosphorylation in these LP 'non-transformed' cells, in which mitochondrial function is reduced. However, pyruvate treatment of transformed LP cells during additional passages did not significantly restore their oxidative metabolism. These data highlight changes accompanying spontaneous astrocyte transformation and suggest potential targets for the control of astrocyte proliferation and reaction to various insults to the central nervous system.

Receptors to steroid hormones and aromatase are expressed by cultured motoneurons but not by glial cells derived from rat embryo spinal cord.

The aim of this study was to examine the expression of aromatase and receptors to steroid hormones in cultured motoneurons (MNs). We first developed an original method for obtaining rat MN cultures. Dissociated E15 rat spinal cords were purified using metrizamide and bovine serum albumin density gradients, and cells were then seeded on the culture substratum. We optimized the culture parameters and found that simple addition of rat muscle extract (ME) and conditioned culture medium (CM) from glial cell lines (GCL) derived from spinal cord were sufficient to obtain almost pure MN cultures. MNs were characterized by the presence of specific MN markers and electrophysiology. MNs could be kept alive for 2 weeks. We demonstrate that ME and CM are essential for MN development and survival respectively. Immunocytochemistry and aromatase activity assay indicated the presence of androgen and estrogen receptors as well as aromatase in MNs but not in GCL. This is the first report demonstrating the presence of both female and male sex hormone receptors and a key enzyme in steroid hormone metabolism in MNs and its absence in GCL, at least in our culture conditions. This in vitro model appears to be valuable for elucidating the impact of the sex hormone circuit in neuronal maturation. The relevance of this model for the comprehension of neurodegenerative diseases is discussed.