An RNA tool kit to study the status of mouse ES cells: sex determination and stemness.

Mouse embryonic stem cells (mESCs) are pluripotent stem cells derived from the inner cell mass of the blastocyst. They can be maintained under controlled culture conditions in a pluripotent state, or be induced to differentiate into all derivatives of the three primary germ layers: ectoderm, endoderm and mesoderm. Several studies have characterised the coding and non-coding (nc) RNA repertoires of mESCs, uncovering highly dynamic variations during the process of differentiation, but also qualitative differences pertaining to sex. For example, up-regulation of the long non-coding RNA Xist on the X chromosome induces gene silencing and X inactivation exclusively during female mESC differentiation. In contrast, specific small RNAs have been shown to be up-regulated during male mESC differentiation. Here, we illustrate how a small set of key coding and ncRNAs can be exploited as dynamic and sensitive markers of the stemness and/or the differentiation status of male or female mESC lines. We describe adapted techniques for the extended characterization and analysis of mESCs from as little material as that cultured in a single 75cm(2) flask.

A SAGE approach to identifying novel trans-acting factors involved in the X inactivation process.

X chromosome inactivation ensures the dosage compensation of X-linked genes in XX females compared to their XY male counterpart. It is characterised by the specific recruitment of an inhibitory ribonucleoprotein complex involving the non-coding Xist RNA to the presumptive inactive X chromosome and associated chromatin modifications, which result in the transcriptional silencing of the X chromosome. As an approach to the identification of some of the potential molecular players in this process we have performed comparative transcriptional profiling of mouse 6.5-dpc (days post-coitum) female and male embryos using a modified SAGE (Serial analysis of gene expression) technique which allows the analysis of small quantities of biological material. At 6.5 dpc, a moment when random X inactivation of embryonic tissues has just been achieved, some two hundred transcripts that were significantly enriched in the female gastrula compared to its male counterpart could be identified. The validation of an association with the X inactivation process of a subset of these transcripts has been studied, ex vivo, in differentiating female and male ES cells and in female ES cells in which the establishment of X inactivation is interrupted through the post-transcriptional inhibition of Xist synthesis.

[Poisoning by beta,beta'-iminodipropionitrile (IDPN) in the rat: an experimental model for elective lesions of peripheral motoneurons]. / Intoxication par le beta-beta'-iminodipropionitrile (IDPN) chez le rat: un modèle expérimental de lésion élective du montoneurone périphérique.

Chronic or acute intoxication of rats with beta,beta'-iminodipropionitrile induces characteristic lesions of axons of anterior horn cells. Swellings of proximal axons are associated with disorganization of the cytoskeleton which includes a disorientation of neurofilaments which are segregated in the periphery of the axon, while microtubules and mitochondria are clustered centrally. Slow axonal transport, which is markedly reduced, results in accumulation of neurofilaments in the proximal part of the peripheral motoneuron and distal atrophy. In chronically intoxicated rats, proliferation of Schwann cells with onion bulb formation were observed in the anterior spinal roots after 10 months on diet. In distal sciatic nerve, axonal degeneration was associated with accumulation of neurofilaments, organelles and glycogen. Axonal regeneration occurred in spite of sustained intoxication. The intensity of the lesions induced by IDPN in the proximal part of the axon of the spinal motoneuron are reminiscent of those observed in degenerative motor neuron disease. However, the abnormalities of the myelin sheath and the proliferation of Schwann cells encountered in IDPN-intoxicated rats do not occur in degenerative motor neuron diseases in humans.

[Crow-Fukase syndrome (POEMS syndrome) and osseous mastocytosis secondary to Castleman's angiofollicular lymphoid hyperplasia]. / Syndrome de Crow-Fukase ("P.O.E.M.S. syndrome") et mastocytose osseuse secondaires à une hyperplasie lymphoïde angiofolliculaire de Castleman.

In a case of Crow-Fukase (POEMS) syndrome there was a chronic, progressive, and eventually lethal polyradiculoneuropathy. In addition, adenomegaly, oedema and pleural effusions, gonadic atrophy, serum monoclonal IgA, and skin pigmentation were present. Plain x-rays and CT scan of the pelvis and lower vertebrae showed multiple poorly defined lesions. At postmortem there was no myeloma and a bone mastocytosis was found. In addition, next to T11, there was an abdominal nodule, 2 cm in diameter, with histological characteristics of Castleman's angiofollicular lymphoid hyperplasia. Immunohistochemical studies showed that plasmocytes of this lesion secreted polyclonal immunoglobulins with a high prevalence of IgA. Thus, the primary interest of this case lies in the association of bone mastocytosis with a POEMS syndrome. Also, one single localisation of Castleman angiofollicular lymphoid hyperplasia was found, assumed to be in this case the cause of the POEMS syndrome. Therefore, a minute and benign hyperplasic lesion, which was only discovered at autopsy, secreted the protein responsible for the symptoms and signs, and eventually the patient's death.

[MRI symptomatology of primary intraspinal cord gliomas]. / Sémiologie IRM des gliomes primitifs intra-médullaires.

MRI has now been recognized as the best technique for exploration of spinal tumours and, in particular, tumours within the spinal cord. Based on a retrospective study of 74 operated glial tumours, we are trying to define a specific semiology for intramedullary astrocytomas and ependymomas. Thirty-four cases were selected including 17 astrocytomas (7 low-grade, 10 high-grade) and 17 ependymomas (1 of which was grade III) for whom the pre-operative MRI examination was complete, with T1-weighted sequences without, then with gadolinium, and T2-weighted sequences. The examination was performed using a high-field and in most cases 1.5 Tesla machine. Analysis, correlated with operative data and pathology results, comprised on the one hand patients' distribution by age, sex and location of the tumour on the spinal cord, and on the other hand the MRI semiology concerning the sagittal and axial localization of the fleshy portion after gadolinium injection, the limits of the tumour, the homo- or heterogeneous character of its enhancement, the possible existence of stigmas of intra- or peritumoral chronic bleeding, and finally the presence or absence of associated cysts in the 34 exploitable cases. Some semiological differences were elicited between astrocytomas and ependymomas: the patient's age at the time of diagnosis was predominantly 0 to 20 for astrocytomas (astrocytomas 39%, ependymomas 4%), and the well-limited character of the fleshy portion of the tumour after gadolinium injection was found in 70% of ependymomas, 40% of high-grade astrocytomas and 14% of low-grade astrocytomas. The homogeneity of contrast enhancement in ependymomas has been classically defined, but it did not show in our series. Finally, it seems that high-grade astrocytomas are characterized by the rare presence of hemosiderin deposits (high-grade 20%, low-grade 57%, ependymomas 58%) and by the absence or reduced extension of overlying and underlying cysts.

Antiviral RNA interference in mammalian cells.

In antiviral RNA interference (RNAi), the DICER enzyme processes virus-derived double-stranded RNA (dsRNA) into small interfering RNAs (siRNAs) that guide ARGONAUTE proteins to silence complementary viral RNA. As a counterdefense, viruses deploy viral suppressors of RNAi (VSRs). Well-established in plants and invertebrates, the existence of antiviral RNAi remains unknown in mammals. Here, we show that undifferentiated mouse cells infected with encephalomyocarditis virus (EMCV) or Nodamura virus (NoV) accumulate ~22-nucleotide RNAs with all the signature features of siRNAs. These derive from viral dsRNA replication intermediates, incorporate into AGO2, are eliminated in Dicer knockout cells, and decrease in abundance upon cell differentiation. Furthermore, genetically ablating a NoV-encoded VSR that antagonizes DICER during authentic infections reduces NoV accumulation, which is rescued in RNAi-deficient mouse cells. We conclude that antiviral RNAi operates in mammalian cells.

The peripheral neuropathy of necrotizing arteritis: a clinicopathological study.

We have reviewed the clinical and morphological data from 100 patients with necrotizing arteritis in muscle and/or in nerve samples taken by biopsy. The neuropathy occurred in the context of a multisystem disorder (Group 1) or in apparent isolation (Group 2). The average age of patients was 59 in Group 1 and 61 in Group 2. Females were more commonly affected than males, especially in the first group. Necrotizing arteritis complicated the course of rheumatoid arthritis in 25 patients. In 3 patients necrotizing arteritis was associated with infection with the human immunodeficiency virus, the agent of AIDS. Tests for hepatitis B surface antigen were positive in 19 patients. Mononeuritis was present in 13, mononeuritis multiplex in 62, and distal symmetrical sensory or sensorimotor neuropathy in 19 patients. In both groups of patients, the muscle biopsy was more frequently diagnostic for arteritis than was the nerve biopsy (80% versus 55%). The average incidence of isolated fibers undergoing axonal degeneration was 64.8%; that of demyelinated/remyelinated fibers was 1.9%. We conclude that the combination of nerve and muscle sampling increases the chance of visualizing characteristic arterial lesions in vasculitic neuropathy.

Low levels of human immunodeficiency virus replication in the brain tissue of children with severe acquired immunodeficiency syndrome encephalopathy.

The authors examined the autopsy brain samples of nine children infected with human immunodeficiency virus (HIV) at birth by histology, immunologic staining, and in situ hybridization. Surprisingly, although seven of these children presented with typical AIDS encephalopathy, the authors could detect a multifocal HIV infection in the brains of only three of these patients. The authors could not detect any significant HIV replication in the brain of four other children despite severe neurologic disease. However, HIV DNA was detected by polymerase chain reaction (PCR) in the central nervous system (CNS) of all patients. In addition, the authors found associated lesions in the brains of three of these four patients. This study shows that severe AIDS encephalopathy exists in children and therefore might exist in adults with few signs or without any signs of HIV replication or inflammation in the CNS. Understanding the pathogenesis of this neurologic disease and the kinetics of HIV replication in brain tissue of children with AIDS encephalopathy is essential to determine the best therapeutic strategy.