Patricia Flamant

Human papillomaviruses associated with cervical intraepithelial neoplasia. Great diversity and distinct distribution in low- and high-grade lesions.

&NA; All together, 30 genital human papillomavirus (HPV) types have been characterized so far. To evaluate the importance of HPV diversity in associated cervical diseases, we analyzed 188 biopsy specimens obtained from patients with a recent diagnosis of cervical HPV infection or intraepithelial neoplasia (CIN). Of these 188 specimens, 116 were classified as low‐grade CIN (48 cases), high‐grade CIN (53 cases), condylomata acuminata (10 cases), flat condylomas (five cases). Seventy‐two specimens were considered nondiagnostic. Using probes specific for 18 genital HPV types, HPV DNA sequences were detected by Southern blot hybridization in 100 lesions and 21 nondiagnostic specimens. When further analyzed by the polymerase chain reaction, eight HPV‐negative biopsy specimens, four CIN, and four nondiagnostic specimens were positive. Of the 129 positive biopsy specimens, 92 contained at least one of 18 known HPV types and 37 HPV that have not yet been identified. Nine specimens had more than one type. Thirteen HPV types were identified in CIN. The detection rate of HPV 16 increased from 21% in low‐grade CIN to 57% in highgrade CIN. HPV 18 was detected in only 3% of CIN; HPV 31, 33, and 35 were found in 8%. HPV 30, 39, 45, 51, 52, 56, 58, and 61 were detected in 44% of low‐grade CIN but in only 8% of high‐grade CIN. Unidentified HPV were detected in about 25% of low‐grade and high‐grade CIN. Fifty‐seven CIN positive for at least one HPV type were further analyzed by in situ hybridization. Thirty‐five (65%) biopsy specimens were positive, including 21 of 24 low‐grade CIN and 14 of 33 high‐grade CIN. Ten of the 13 previously identified HPV types were detected. Thus, CIN represents an heterogeneous disease from a virologic viewpoint. This fact could explain their variable clinical evolution.

Variation in the Nucleotide Sequence of Cottontail Rabbit Papillomavirus a and b Subtypes Affects Wart Regression and Malignant Transformation and Level of Viral Replication in Domestic Rabbits

ABSTRACT We previously reported the partial characterization of two cottontail rabbit papillomavirus (CRPV) subtypes with strikingly divergent E6 and E7 oncoproteins. We report now the complete nucleotide sequences of these subtypes, referred to as CRPVa4 (7,868 nucleotides) and CRPVb (7,867 nucleotides). The CRPVa4 and CRPVb genomes differed at 238 (3%) nucleotide positions, whereas CRPVa4 and the prototype CRPV differed by only 5 nucleotides. The most variable region (7% nucleotide divergence) included the long regulatory region (LRR) and the E6 and E7 genes. A mutation in the stop codon resulted in an 8-amino-acid-longer CRPVb E4 protein, and a nucleotide deletion reduced the coding capacity of the E5 gene from 101 to 25 amino acids. In domestic rabbits homozygous for a specific haplotype of the DRA and DQA genes of the major histocompatibility complex, warts induced by CRPVb DNA or a chimeric genome containing the CRPVb LRR/E6/E7 region showed an early regression, whereas warts induced by CRPVa4 or a chimeric genome containing the CRPVa4 LRR/E6/E7 region persisted and evolved into carcinomas. In contrast, most CRPVa, CRPVb, and chimeric CRPV DNA-induced warts showed no early regression in rabbits homozygous for another DRA-DQA haplotype. Little, if any, viral replication is usually observed in domestic rabbit warts. When warts induced by CRPVa and CRPVb virions and DNA were compared, the number of cells positive for viral DNA or capsid antigens was found to be greater by 1 order of magnitude for specimens induced by CRPVb. Thus, both sequence variation in the LRR/E6/E7 region and the genetic constitution of the host influence the expression of the oncogenic potential of CRPV. Furthermore, intratype variation may overcome to some extent the host restriction of CRPV replication in domestic rabbits.

More efficient repair of DNA double-strand breaks in skeletal muscle stem cells compared to their committed progeny.

The loss of genome integrity in adult stem cells results in accelerated tissue aging and is possibly cancerogenic. Adult stem cells in different tissues appear to react robustly to DNA damage. We report that adult skeletal stem (satellite) cells do not primarily respond to radiation-induced DNA double-strand breaks (DSBs) via differentiation and exhibit less apoptosis compared to other myogenic cells. Satellite cells repair these DNA lesions more efficiently than their committed progeny. Importantly, non-proliferating satellite cells and post-mitotic nuclei in the fiber exhibit dramatically distinct repair efficiencies. Altogether, reduction of the repair capacity appears to be more a function of differentiation than of the proliferation status of the muscle cell. Notably, satellite cells retain a high efficiency of DSB repair also when isolated from the natural niche. Finally, we show that repair of DSB substrates is not only very efficient but, surprisingly, also very accurate in satellite cells and that accurate repair depends on the key non-homologous end-joining factor DNA-PKcs.

Numb is required to prevent p53-dependent senescence following skeletal muscle injury

Regeneration relies on coordinated action of multiple cell types to reconstitute the damaged tissue. Here we inactivate the endocytic adaptor protein Numb in skeletal muscle stem cells prior to chronic or severe muscle injury in mice. We observe two types of senescence in regenerating muscle; a transient senescence in non-myogenic cells of control and Numb mutant mice that partly depends on INK4a/ARF activity, and a persistent senescence in myogenic cells lacking Numb. The senescence levels of Numb-deficient muscle is reduced to wild type levels by an anti-oxidant treatment or p53 ablation, resulting in functional rescue of the regenerative potential in Numb mutants. Ex vivo experiments suggest that Numb-deficient senescent cells recruit macrophages to sustain inflammation and drive fibrosis, two hallmarks of the impaired muscle regeneration in Numb mutants. These findings provide insights into previously reported developmental and oncogenic senescence that are also differentially regulated by p53.

GM130 gain-of-function induces cell pathology in a model of lysosomal storage disease

Cell pathology in lysosomal storage diseases is characterized by the formation of distended vacuoles with characteristics of lysosomes. Our previous studies in mucopolysaccharidosis type IIIB (MPSIIIB), a disease in which a genetic defect induces the accumulation of undigested heparan sulfate (HS) fragments, led to the hypothesis that abnormal lysosome formation was related to events occurring at the Golgi level. We reproduced the enzyme defect of MPSIIIB in HeLa cells using tetracycline-inducible expression of shRNAs directed against α-N-acetylglucosaminidase (NAGLU) and addressed this hypothesis. HeLa cells deprived of NAGLU accumulated abnormal lysosomes. The Golgi matrix protein GM130 was over-expressed. The cis- and medial-Golgi compartments were distended, elongated and formed circularized ribbons. The Golgi microtubule network was enlarged with increased amounts of AKAP450, a partner of GM130 controlling this network. GM130 down-regulation prevented pathology in HeLa cells deprived of NAGLU, whereas GM130 over-expression in control HeLa cells mimicked the pathology of deprived cells. We concluded that abnormal lysosomes forming in cells accumulating HS fragments were the consequence of GM130 gain-of-function and subsequent alterations of the Golgi ribbon architecture. These results indicate that GM130 functions are modulated by HS glycosaminoglycans and therefore possibly controlled by extracellular cues.

Phenotypic clustering: a novel method for microglial morphology analysis

BackgroundMicroglial cells are tissue-resident macrophages of the central nervous system. They are extremely dynamic, sensitive to their microenvironment and present a characteristic complex and heterogeneous morphology and distribution within the brain tissue. Many experimental clues highlight a strong link between their morphology and their function in response to aggression. However, due to their complex “dendritic-like” aspect that constitutes the major pool of murine microglial cells and their dense network, precise and powerful morphological studies are not easy to realize and complicate correlation with molecular or clinical parameters.MethodsUsing the knock-in mouse model CX3CR1GFP/+, we developed a 3D automated confocal tissue imaging system coupled with morphological modelling of many thousands of microglial cells revealing precise and quantitative assessment of major cell features: cell density, cell body area, cytoplasm area and number of primary, secondary and tertiary processes. We determined two morphological criteria that are the complexity index (CI) and the covered environment area (CEA) allowing an innovative approach lying in (i) an accurate and objective study of morphological changes in healthy or pathological condition, (ii) an in situ mapping of the microglial distribution in different neuroanatomical regions and (iii) a study of the clustering of numerous cells, allowing us to discriminate different sub-populations.ResultsOur results on more than 20,000 cells by condition confirm at baseline a regional heterogeneity of the microglial distribution and phenotype that persists after induction of neuroinflammation by systemic injection of lipopolysaccharide (LPS). Using clustering analysis, we highlight that, at resting state, microglial cells are distributed in four microglial sub-populations defined by their CI and CEA with a regional pattern and a specific behaviour after challenge.ConclusionsOur results counteract the classical view of a homogenous regional resting state of the microglial cells within the brain. Microglial cells are distributed in different defined sub-populations that present specific behaviour after pathological challenge, allowing postulating for a cellular and functional specialization. Moreover, this new experimental approach will provide a support not only to neuropathological diagnosis but also to study microglial function in various disease models while reducing the number of animals needed to approach the international ethical statements.

New in vivo avatars of diffuse intrinsic pontine gliomas (DIPG) from stereotactic biopsies performed at diagnosis

Diffuse Instrinsic Pontine Glioma is the most aggressive form of High Grade Gliomas in children. The lack of biological material and the absence of relevant models have hampered the development of new therapeutics. Their extensive infiltration of the brainstem renders any surgical resection impossible and until recently biopsies were considered not informative enough and therefore not recommended. Thus, most models were derived from autopsy material. We aimed to develop relevant in vivo DIPG models that mimic this specific disease and its molecular diversity from tumor material obtained at diagnosis. Eight patient-derived orthotopic xenograft models were obtained after direct stereotactic injection of a mixed cell suspension containing tumor cells and stromal cells in the brainstem or thalamus of nude mice and serially passaged thereafter. In parallel, we developed 6 cell-derived xenograft models after orthotopic injection of tumor-initiating cells cultured from stereotactic biopsies. Cells were modified to express luciferase to enable longitudinal tumor growth monitoring, and fluorescent reporter proteins to trace the tumor cells in the brain. These models do not form a tumor mass, they are invasive, show the H3K27 trimethylation loss in vivo and the tumor type diversity observed in patients in terms of histone H3 mutations and lineage markers. Histological and MRI features at 11.7 Tesla show similarities with treatment naïve human DIPG, and in this respect, both direct and indirect orthotopic xenograft looked alike. These DIPG models will therefore constitute valuable tools for evaluating new therapeutic approaches in this devastating disease.Diffuse Instrinsic Pontine Glioma is the most aggressive form of High Grade Gliomas in children. The lack of biological material and the absence of relevant models have hampered the development of new therapeutics. Their extensive infiltration of the brainstem renders any surgical resection impossible and until recently biopsies were considered not informative enough and therefore not recommended. Thus, most models were derived from autopsy material. We aimed to develop relevant in vivo DIPG models that mimic this specific disease and its molecular diversity from tumor material obtained at diagnosis. Eight patient-derived orthotopic xenograft models were obtained after direct stereotactic injection of a mixed cell suspension containing tumor cells and stromal cells in the brainstem or thalamus of nude mice and serially passaged thereafter. In parallel, we developed 6 cell-derived xenograft models after orthotopic injection of tumor-initiating cells cultured from stereotactic biopsies. Cells were modified to express luciferase to enable longitudinal tumor growth monitoring, and fluorescent reporter proteins to trace the tumor cells in the brain.These models do not form a tumor mass, they are invasive, show the H3K27 trimethylation loss in vivo and the tumor type diversity observed in patients in terms of histone H3 mutations and lineage markers. Histological and MRI features at 11.7 Tesla show similarities with treatment naïve human DIPG, and in this respect, both direct and indirect orthotopic xenograft looked alike. These DIPG models will therefore constitute valuable tools for evaluating new therapeutic approaches in this devastating disease.