Impact of the hypoxic microenvironment on spermatogonial stem cells in culture.

The stem cell niche plays a crucial role in the decision to either self-renew or differentiate. Recent observations lead to the hypothesis that O2 supply by blood and local O2 tension could be key components of the testicular niche of spermatogonial stem cells (SSCs). In this study, we investigated the impact of different hypoxic conditions (3.5%, 1%, and 0.1% O2 tension) on murine and human SSCs in culture. We observed a deleterious effect of severe hypoxia (1% O2 and 0.1% O2) on the capacity of murine SSCs to form germ cell clusters when plated at low density. Severe effects on SSCs proliferation occur at an O2 tension ≤1% and hypoxia was shown to induce a slight differentiation bias under 1% and 0.1% O2 conditions. Exposure to hypoxia did not appear to change the mitochondrial mass and the potential of membrane of mitochondria in SSCs, but induced the generation of mitochondrial ROS at 3.5% and 1% O2. In 3.5% O2 conditions, the capacity of SSCs to form colonies was maintained at the level of 21% O2 at low cell density, but it was impossible to amplify and maintain stem cell number in high cell density culture. In addition, we observed that 3.5% hypoxia did not improve the maintenance and propagation of human SSCs. Finally, our data tend to show that the transcription factors HIF-1α and HIF-2α are not involved in the SSCs cell autonomous response to hypoxia.

Noninvasive Assessment of Neurodevelopmental Disorders after In Utero Irradiation in Mice: An In Vivo Anatomical and Diffusion MRI Study.

In utero exposure to ionizing radiation can lead to cerebral alterations during adulthood. Using anatomical magnetic resonance imaging (MRI), it is possible to assess radiation-induced structural brain damage noninvasively. However, little is currently known about microstructure alterations in brain tissue. Therefore, the goal of this study was to establish, based on an original and robust pipeline of MRI image analysis, whether the long-term effects of in utero radiation exposure on brain tissue microstructure could be detected noninvasively. Pregnant C57BL/6N mice received a single dose of 1 Gy on gestation day 14.5, which led to behavioral impairments in adults. At 3 months old, in vivo MRI data were acquired from in utero irradiated and nonirradiated male mice. An MRI protocol was designed to assess the effects of radiation on the parameters of brain volume, non-Gaussian diffusion (ADC0, kurtosis and signature index) and anisotropic diffusion (fractional anisotropy and mean, axial, radial diffusivities and anisotropic signature index) in 10 key cerebral structures defined using an in-house atlas of the mouse brain. Based on the relative amplitude of these anatomical and microstructural changes, maps of the radiosensitivity of the brain to in utero irradiation were created. We observed microcephaly in irradiated mice with noticeably larger volume changes in the cortex and the corpus callosum. We also observed significantly lower ADC0, anisotropy fraction (sFA), radial diffusivity (sRD), as well as signature index (S-index and SI3) values, which are original markers sensitive to tissue microstructure alterations. All these changes together are in favor of a decreased cellular "imprint" and in some regions a reduced density in myelinated axons. A reduction in the number and complexity of myelinated axons was further revealed by myelin basic protein immunostaining. Combining anatomical and diffusion MRI is a promising approach to noninvasively investigate the radiosensitivity of local brain areas in adult mice after in utero irradiation in terms of microstructure.

Telomere dynamics, end-to-end fusions and telomerase activation during the human fibroblast immortalization process.

Loss of telomeric repeats during cell proliferation could play a role in senescence. It has been generally assumed that activation of telomerase prevents further telomere shortening and is essential for cell immortalization. In this study, we performed a detailed cytogenetic and molecular characterization of four SV40 transformed human fibroblastic cell lines by regularly monitoring the size distribution of terminal restriction fragments, telomerase activity and the associated chromosomal instability throughout immortalization. The mean TRF lengths progressively decreased in pre-crisis cells during the lifespan of the cultures. At crisis, telomeres reached a critical size, different among the cell lines, contributing to the peak of dicentric chromosomes, which resulted mostly from telomeric associations. We observed a direct correlation between short telomere length at crisis and chromosomal instability. In two immortal cell lines, although telomerase was detected, mean telomere length still continued to decrease whereas the number of dicentric chromosomes associated was stabilized. Thus telomerase could protect specifically telomeres which have reached a critical size against end-to-end dicentrics, while long telomeres continue to decrease, although at a slower rate as before crisis. This suggests a balance between elongation by telomerase and telomere shortening, towards a stabilized 'optimal' length.

Fibroblast growth factor 2 up regulates telomerase activity in neural precursor cells.

During brain development, neuronal and glial cells are generated from neural precursors on a precise schedule involving steps of proliferation, fate commitment and differentiation. We report that telomerase activity is highly expressed during embryonic murine cortical neurogenesis and early steps of gliogenesis and progressively decreases thereafter during cortex maturation to be undetectable in the normal adult brain. We evidenced neural precursor cells (NPC) as the principal telomerase-expressing cells in primary cultures from E15 mouse embryo cortices. Their differentiation either in neurons or in glial cells lead to a down regulation of telomerase activity that was directly correlated to the decrease of telomerase core protein (mTERT) mRNA synthesis. Furthermore, we show that FGF2 (fibroblast growth factor 2), one of the main regulators of CNS development, induces a dose-dependant increase of both the proliferation of NPC and telomerase activity in primary cortical cultures without affecting the mTERT mRNA synthesis compared to that of glyceraldehyde-3-phosphate dehydrogenase (mGAPDH). Finally, we evidenced that AZT (3'-azido-2', 3'-dideoxythymidine), known to inhibit telomerase activity, blocks in a dose dependant manner the FGF2-induced proliferation of NPC. Altogether, our results are in favor of an important role of telomerase activity during brain organogenesis. Oncogene (2000).

Molecular detection of telomerase-positive circulating epithelial cells in metastatic breast cancer patients.

The detection of circulating tumor cells and micrometastases may have important therapeutic and prognostic implications. Telomerase is a hallmark of cancer and is absent from normal epithelial cells. The aim of this study was to use telomerase activity as a molecular marker for the detection of cancer cells in blood of patients with breast cancer. Blood samples were collected from 25 women with stage IV breast cancer and 9 healthy volunteers. Peripheral blood mononuclear cells were isolated by using Ficoll/Hypaque. Immunomagnetic beads coated with an epithelial-specific antibody (BerEP4) were used to harvest epithelial cells from peripheral blood mononuclear cells. Telomerase activity was detected in harvested epithelial cells (HECs) using two different telomerase-PCR-ELISA methods. HECs from blood samples of 21 of 25 (84%) patients with breast cancer were telomerase positive. Telomerase activity was undetectable in HECs from the nine healthy volunteers, demonstrating the specificity of the association between telomerase activity in HECs and stage IV breast cancer. Thus, determination of telomerase activity in HECs appears to be a sensitive, specific, and noninvasive approach for detecting circulating epithelial cancer cells in patients with metastatic breast cancer. This method could be of great value in monitoring the cancer cell proliferation during chemotherapy. This study should be now extended to patients with early-stage breast cancer to investigate the role of telomerase expression by HECs and to evaluate its prognostic value.

Correlation between HIV provirus burden and in utero transmission.

OBJECTIVE: No predictive parameters of in utero or perinatal vertical transmission of HIV to newborns are known at present. Vertical transmission may be related to several biological parameters of maternal HIV infection: (1) immunological parameters (neutralizing antibodies); (2) the concentration of viral particles and/or infected cells; and (3) the selection of HIV subspecies of particular cellular tropism. The present study was designed to examine the relationship between cellular viral burden and transmission, and between maternal viral burden and CD4+ cell count and clinical status at delivery. METHOD: We investigated mother-to-infant HIV-1 transmission at delivery in a cohort of 51 pairs of mothers and newborns. Twelve infants were HIV-infected, as determined by successive polymerase chain reaction and culture determinations within the first 6 months of life, and nine of these were diagnosed as HIV-infected during the first week of life. We determined peripheral blood mononuclear cell proviral DNA burden using a quantitative polymerase chain reaction assay. Polymerase chain reaction was performed in the HIV-1 gag gene, using [32P]-end-labelled primers. External standard DNA samples were from the 85-14 F2 cell line, which contains a unique defective proviral DNA genome. RESULTS: There was a linear relationship between the logarithms of c.p.m. and the number of HIV-1 DNA copies. CONCLUSION: We have previously reported that the number of HIV provirus copies in maternal blood cells is related to transmission of the virus. Quantification of the HIV provirus by polymerase chain reaction may be used as a predictive parameter of vertical transmission if accompanied by an exhaustive clinical and biological follow-up during pregnancy.

Interleukin 1 beta, interleukin 6, tumor necrosis factor alpha, and interleukin 10 responses in peripheral blood mononuclear cells of cynomolgus macaques during acute infection with SIVmac251.

HIV infection ultimately leads to AIDS, despite the immune responses elicited soon after infection. In addition to the observed changes in lymphoid cell subsets, alteration of the cytokine network most likely accompanies and/or contributes to the lack of protective immune responses. In an attempt to delineate the early events in the immune response to lentivirus infection, we have sequentially monitored levels of proinflammatory (IL-1 beta, IL-6, and TNF-alpha) and antiinflammatory (IL-10) cytokine mRNAs in PBMCs of cynomolgus macaques during primary SIVmac infection. Eight monkeys were infected i.v. with 4 AID50 of cell-free SIVmac251. All monkeys seroconverted between days 16 and 21 postinfection (p.i.), and had detectable peripheral viremia. The viral load peaked between days 12 and 16 p.i., and fell sharply thereafter. A marked increase in the expression of IL-6 mRNA was observed in all macaques during the first weeks following infection. An increase in the levels of expression of IL-1 beta, TNF-alpha, and IL-10 mRNA was also determined in six, six, and five of the eight monkeys, respectively. While IL-6, TNF-alpha, and IL-10 increased transiently, increased levels of IL-1 beta mRNA expression were sustained over 44 days in most monkeys.

A simple and rapid method for sequencing the entire coding region of the human prion protein (PrP) gene.

The accumulation in brain of the 'prion protein' (PrP), a host-encoded sialoglycoprotein, is the unique specific molecular marker of subacute spongiform transmissible encephalopathies (SSTE). Furthermore, the primary sequence of the PrP gene (PRNP) seems to contain some genetic determinants of great importance in the development of SSTE. Here we present a simple and rapid polymerase chain reaction (PCR)-based method for direct sequencing of the entire coding sequence of the PrP gene, PRNP, in patients. The ability to determine sequences of both alleles of the PRNP gene is demonstrated in the analysis of 3 patients previously established as codon 129 heterozygotes by the use allele-specific oligonucleotide hybridization method.

Astrocyte gene expression in experimental mouse scrapie.

The biological hallmark of transmissible spongiform encephalopathies is a significant accumulation, in brain, of the scrapie prion protein (PrPsc), often associated with an increased glial fibrillary acidic protein (GFAP) expression. This study was focused on astrocyte gene expression during scrapie development over a period of 172 days in intracerebrally inoculated newborn mice. The levels of expression of PrP and two specific astrocyte proteins, -GFAP and glutamine synthetase (GS)-, were investigated by Western and Northern blots. In brain, a 10-fold increased expression of GFAP mRNAS was demonstrated from 112 days post-inoculation to 172 days, whereas the "upregulation" of GS mRNAs was two-fold. GFAP was observed to increase 10- to 20-fold in scrapie-infected brain from day 112 to day 172, while PrP showed a three- to four-fold elevation. Both proteins were found in greater amount in the frontal cortex and cerebellum of animals with clinical scrapie than in those given an injection of normal brain. PrPsc was detected in scrapie brain from day 84 after inoculation, and thereafter increased about 20-fold until day 172. On the other hand, the concentration of glutamine synthetase remained constant in brain throughout the scrapie disease. To conclude, these results show that GFAP and GS mRNAs are differently upregulated in brain in the scrapie mouse model.

Radiation-induced upregulation of telomerase activity escapes PI3-kinase inhibition in two malignant glioma cell lines.

Tumor relapse after radiotherapy is a great concern in the treatment of high-grade gliomas. Inhibition of the PI3-kinase/AKT pathway is known to radiosensitize cancer cells and to delay their DNA repair after irradiation. In this study, we show that the radiosensitization of CB193 and T98G, two high-grade glioma cell lines, by the PI3K inhibitor LY294002, correlates with the induction of G1 and G2/M arrest, but is inconsistently linked to a delayed DNA double-strand break (DSBs) repair. The PI3K/AKT pathway has been shown to activate radioprotective factors such as telomerase, whose inhibition may contribute to the radiosensitization of cancer cells. However, we show that radiation upregulates telomerase activity in LY-294002-treated glioma cells as well as untreated controls, demonstrating a PI3K/AKT-independent pathway of telomerase activation. Our study suggests that radiosensitizing strategies based on PI3-kinase inhibition in high-grade gliomas may be optimized by additional treatments targeting either telomerase activity or telomere maintenance.

[Interactions between HIV virus and the nervous system: recent pathogenic data and hypotheses]. / Interactions virus VIH et système nerveux: données récentes et hypothèses pathogéniques.

Disorders of the nervous system frequently complicate Acquired Immune Deficiency Syndrome (AIDS). They may be related to the development of opportunistic agents (toxoplasmosis, cryptococcossis, cytomegalovirus, JC Virus), or primary CNS lymphoma. There is also a constellation of neurologic disorders which may result from direct Human Immunodeficiency Virus (HIV) replication in the CNS and HIV has been found in brain and CSF of numerous patients suffering from AIDS. The precise cellular localization of HIV is not known, but the macrophage seems to be a strong candidate for HIV replication in CNS.

[Isolation, culture and characterization of astrocytes from adult monkey brains]. / Isolement, culture et caractérisation d'astrocytes issus de cerveaux de singes adultes.

We have developed protocols for the isolation, the culture and the immunocytochemical characterization of astrocytes from simian adult brains. We have obtained pure astrocyte cultures without contamination with other cells present in brain. No microglial cells, oligodendrocytes, neurons or fibroblasts were detected by specific staining in immunocytochemistry.

Inactivation of human immunodeficiency virus during human growth hormone production.

Since human immunodeficiency virus (HIV) has been found in the brains of AIDS patients, the possibility was investigated that preparations of human growth hormone (hGH) extracted from human pituitary glands harbor infectious HIV. It was found that the procedure used for extracting hGH, i.e. a combination of acid pH and 10% ethanol, totally inactivates HIV infectivity.

Mechanisms of downmodulation and release of tumour necrosis factor receptor induced by human immunodeficiency virus type 1 in human monocytes.

Infection of human monocytes with human immunodeficiency virus type (HIV-1 LAI) triggers the release of both the cytokine tumour necrosis factor alpha (TNF-alpha) and its soluble receptor (TNFsr). In the present study, the authors have investigated the cellular events implicated in the modulation of expression and shedding of the monocyte TNF receptor induced by HIV-1 LAI. Release of TNFsr75 was triggered at an early step of interaction of the virus particles with the monocyte, involving the envelope glycoprotein gp120. HIV-1 LAI induced an upregulation of TNFr75 mRNA, whereas TNFr55 mRNA was not detectable. TNFsr75 release required exocytosis, proteolytic cleavage by serine protease(s), but was independent of prior endocytosis of the receptor. Early shedding of TNFr75 accounted for the almost total but transient disappearance of the membrane TNF receptor P75, observed 60 min after activation with HIV-1 LAI, whereas internalization was minimal. Endogenous TNF-alpha had no role in the disappearance of its own receptor. Complete and stable restoration of TNFr expression at the cell membrane, dependent on de novo protein synthesis, occurred after 5 h, followed by massive TNFsr75 release. These results demonstrate that interaction of human monocytes with HIV-1 LAI triggers at an early stage a cascade of cellular events that lead to profound remodeling of the cell TNFr pool. Understanding the mechanisms of these receptor movements is of importance to document the central role of the TNF system in HIV infection.

Granulocyte macrophage colony stimulating factor stimulates in vitro proliferation of astrocytes derived from simian mature brains.

In the brain, granulocyte-macrophage colony stimulating factor (GM-CSF) may be released by infiltrated cells of the immune system including T and B lymphocytes and mononuclear phagocytes, but also by nervous system resident cells such as microglia and astrocytes. Astrocyte-secreted GM-CSF may play an important role in enhancing the local inflammatory response to central nervous system (CNS) injury and in recruting microglia and activated macrophages. In this study, we demonstrated that GM-CSF, as TNF alpha and IL 6, stimulates in vitro proliferation of simian astrocytes in primary cultures. Results were confirmed by blocking experiments performed with a specific neutralizing mAb directed against GM-CSF. Furthermore, we demonstrated that GM-CSF mediates its effect on these cells through the alpha subunit of the GM-CSF receptor which is constitutively expressed at the membrane of the cultured simian astrocytes as assessed by immunofluorescence. GM-CSF effects on astrocytes could be involved in astrocytosis, a hallmark of various neurological injuries and in inflammatory processes in an autocrine manner.

Isolation of HIV in a seronegative demented patient without symptoms of immune deficiency.

A 60-year-old male patient, originating from West Africa, developed acute and regressive neurologic symptoms associated with aphasia, apraxia, acalculia, behavioral impairments, and an epileptic phase. Eighteen months after the onset of the disease, the patient was almost normal. All along the clinical course, biological abnormality patterns were minor. We noted only a mild neutropenia in the blood. We also observed a weak lymphocytosis and elevated protein content in the cerebrospinal fluid. Electroencephalogram examination revealed slow waves which disappeared after remission. A weak ventricular dilatation was detected on CT scan. Neither vascular, nor tumoral, nor a classical infectious origin could be identified. While the patient was seronegative to HIV, a HIV-like virus was isolated twice from his peripheral blood lymphocytes during the disease. Eighteen months later, the patient remained seronegative. He developed neither AIDS nor immunodeficiency. The subtype of HIV has been isolated and characterized, and its neurotropism is being investigated.

Simian immunodeficiency virus mac251 infection of astrocytes.

Human immunodeficiency virus type 1 (HIV-1) infection of astrocytes has been demonstrated in the brains of patients with AIDS dementia complex (ADC) and may play an important role in neuropathological pathways of HIV-related encephalopathy. SIVmac-infected monkeys develop an acquired immunodeficiency syndrome (AIDS) with CNS involvement which is quite similar to that seen in human AIDS. We investigated the in vitro infection of primary astrocytes derived from adult macaques with SIVmac251 or an isogenic virus that expresses a non-functional Nef protein (SIVmac251-DeltaNef). In both cases we observed that viral expression was mostly limited to early regulatory genes after a transient phase of late viral gene expression (i.e. env and gag), as reported for HIV-1-infected astrocytes in vivo. Late viral gene expression could be reactivated by TNF-alpha, GM-CSF and IFN-gamma treatment of SIVmac251-infected astrocytes but not by similarly treated SIVmac251-DeltaNef-infected cells. Our findings suggest that Nef is not involved in the restricted expression of SIV in astrocytes, but may be important for astrocytes to function as a viral reservoir in the CNS. In additional experiments, we demonstrated Rev and Nef expression in 17 of 27 primary astrocyte cultures derived from macaques infected by SIVmac251. Nef was located in the cytoplasm of astrocytes infected by SIVmac251 in vivo, but displayed perinuclear localisation after infection in vitro. Attempts to activate late viral gene expression by astrocytes infected in vivo using cytokines or by coculture with human cord blood mononuclear cells were unsuccessful.

DNA G-quadruplexes, telomere-specific proteins and telomere-associated enzymes as potential targets for new anticancer drugs.

Telomeres and telomerase have been subjects to a tremendous attention from scientists and oncologists during the past 5 years. This interest has been motivated by the potential of telomerase as a tumor marker for the diagnosis and the prognosis of cancer. The possible use of telomerase or telomeres as new targets for anticancer drugs also triggered investigations. The expression of telomerase was found in overall 85% of cancers. Telomerase is early expressed during oncogenesis with a gradient indicating that a high level of telomerase expression could be associated with a bad prognosis. Therefore, drugs targeting telomerase and telomeres might be useful in many human tumors with little restrictions regarding the tumor type or on the stage of the disease. Moreover, since telomerase is not or slightly expressed in normal cells, it has been postulated that drugs targeting telomerase would induce low toxicity. The race for the discovery of telomerase inhibitors has started while the identification of the components controlling telomerase, telomeres, cell survival, senescence, and apoptosis was still in progress. The recent identification of components regulating telomere length and telomerase expression (TRF1, TRF2, and tankyrase) opened a variety of new opportunities to control telomerase/telomere interactions. Meanwhile, a proof of principle was provided that changing telomere interactions with telomere binding proteins by chemical or biological means can induce cancer cell death. Interestingly, recent data challenge the old paradigm which suggested that a long exposure to telomerase and telomere inhibitors is necessary to induce anticancer effects. In this paper, we review the most recent information concerning the regulation of telomere length and telomerase expression, with emphasis on mechanisms that might translate into new drug discovery.