Ex vivo model of congenital cytomegalovirus infection and new combination therapies.

INTRODUCTION: Congenital human cytomegalovirus (HCMV) infection is a major public health problem due to severe sequelae in the fetus and newborns. Currently, due to their toxicity anti-CMV treatments cannot be administered to pregnant women. We thus developed an ex vivo model of 1(st) trimester placental CMV infection to observe the route of infection across the placenta and to test the efficacy of various new drugs targeting different stages of viral cycle. METHODS: After validation of the viability of floating villi explants by ELISA ß-HCG, the kinetics of placental infection were determined by immunochemistry and qPCR in this ex vivo model. Antiviral susceptibility was determined in vitro using focus reduction assay and by qPCR in the ex vivo model. RESULTS: The ex vivo model showed viral infection in trophoblasts and mesenchymal space of floating villi. In vitro, antiviral combinations of maribavir with baïcalein or artesunate inhibited viral infection by more than 90%. On the other hand, in ex vivo model, infection was reduced by 40% in presence of maribavir and artesunate. The synergistic effect observed in vitro was not observed ex vivo. DISCUSSION: This model allowed us to understand the CMV spread in 1(st) trimester floating villi better and to analyze the anti-CMV efficacy and toxicity of new drugs that could be administered to pregnant women, either alone or in combination. CONCLUSIONS: Such an ex vivo model could be applied to other viruses such as rubella or parvovirus B19 and in new drug development.

Familial 18 centromere variant resulting in difficulties in interpreting prenatal interphase FISH.

We report here on a familial case of centromeric heteromorphism of chromosome 18 detected by prenatal interphase fluorescence in situ hybridization (FISH) analysis transmitted by the mother to her fetus, and resulting in complete loss of one 18 signal. The prenatal diagnosis was performed by interphase FISH (AneuVysion probe set, and LSI DiGeorge 22q11.2 kit) because of the presence of an isolated fetal cardiac abnormality, and was first difficult to interpret: only one centromeric 18 signal was detectable on prenatal interphase nuclei, along with one signal for the Y and one for the X chromosome. The LSI DiGeorge 22q11.2 kit also showed the absence of one TUPLE 1 signal on all examined nuclei. In fact, the FISH performed on maternal buccal smear displayed the same absence of one chromosome 18 centromeric signal, combined with the presence of two TUPLE1 signals. All these results led to the diagnosis of an isolated 22q11.2 fetal microdeletion that was confirmed on metaphases spreads. This case illustrates once again that the locus specific (LSI) probes are more effective than the alpha centromeric probes for interphase analysis. The development of high-quality LSI probes for chromosomes 18, X and Y could avoid the misinterpretation of prenatal interphase FISH leading to numerous additional and expensive investigations.

Lithium down-regulates tau in cultured cortical neurons: a possible mechanism of neuroprotection.

In tauopathies such as Alzheimer's disease (AD), the moleccular mechanisms of tau protein agregation into neurofibrillary tangles (NFTs) and their contribution to neurodegeneration are not fully understood. Recent studies indirectly demonstrated that tau, regardless of its aggregation, might represent a key mediator of neurodegeneration, especially that induced by the amyloid (Abeta) pathology. Lithium is a medication for bipolar mood disorders. Its therapeutic mechanism of action remains unclear, in part because of the large number of biochemical effects attributed to lithium. Since lithium directly inhibits glycogen synthase kinase-3beta (GSK3beta), a key enzyme involved in tau phosphorylation, it was suggested that the therapeutic use of lithium could be expanded from mood disorders to neurodegenerative conditions. Lithium has been also reported to protect cultured neurons against Abeta toxicity, and to prevent NFTs accumulation and cognitive impairments in transgenic models of tauopathies. However, the exact mechanism of neuroprotection provided by lithium remains unknown. Here, we show that exposure of cultured cortical neurons to lithium decreased tau protein levels. This decrease was not linked to the activation of proteolytic processes including calpains, caspases and proteasome or to neuronal loss, but was rather associated with a reduction in tau mRNA levels. Moreover, prior exposure to lithium, at concentrations effective in reducing tau protein levels, markedly reduced pre-aggregated Abeta-induced neuronal apoptosis. Our findings raise the possibility that lithium could exert its neuroprotective effect against Abeta toxicity through the downregulation of tau proteins and that, at least, by acting at the level of tau mRNA.

Baicalein protects cortical neurons from beta-amyloid (25-35) induced toxicity.

Accumulation of amyloid beta peptide (Abeta) has been suggested to contribute to neurodegeneration in Alzheimer's disease (AD). Since chronic inflammation occurs in AD pathogenesis and lipoxygenases are important mediators of inflammatory processes, we evaluated the effect of lipoxygenase inhibitors on apoptosis induced by Abeta on rat cortical cells. The 12-lipoxygenase inhibitor baicalein attenuated both neuronal apoptosis and c-jun protein over-expression induced by Abeta(25- 35), whereas no protection was found with the broad spectrum lipoxygenase inhibitor nordihydroguaiaretic acid or the 5-lipoxygenase inhibitor caffeic acid. These results suggest that 12-lipoxygenase participates in a c-jun-dependent apoptosis pathway triggered by Abeta(25-35), and that specific 12-lipoxygenase inhibitors might be of interest in AD.

Markers of apoptosis and models of programmed cell death in Alzheimer's disease.

Alzheimer's disease (AD) is neuropathologically marked by the presence of senile plaques composed of beta-amyloid peptide and by neurofibrillary tangles formed by abnormally phosphorylated tau protein. Many authors have also reported a neuronal loss in affected regions of the brain in AD patients. This neuronal degeneration could be linked to the triggering of intracellular pathways leading to apoptosis. Previous works were focused on the links between neuronal apoptosis and tau and amyloid precursor protein (APP) metabolisms. We have analyzed tau gene expression in primary neuronal cultures submitted to an apoptotic stress produced by excitotoxicity or serum deprivation. Glutamate induces an enhancement of tau gene expression in resistant neurons whereas a reduced expression is noted in apoptotic cells. This decrease is similar to what is observed after trophic support withdrawal in neuronal cultures. Neurons expressing phosphorylated tau are more resistant to experimental apoptosis than neurons positively labeled for dephosphorylated tau protein (AT8/Tau 1 epitope). In vitro apoptotic neurons are able to produce membrane blebbings (strongly immunopositive for APP and amyloidogenic fragments) that are secondary released in the extracellular space. Finally neurons overexpressing human mutated presenilin 1 (M146 L) are more prone to degenerate than neurons overexpressing human wild-type presenilin 1 after apoptosis induction.

N-methyl-D-aspartate receptor blockade enhances neuronal apoptosis induced by serum deprivation.

Neuronal apoptosis a hallmark of brain development could also be involved in neurodegenerative diseases. Glutamate toxicity is widely proposed as an important factor in the pathogenesis of neurological disorders. We show here that, in rat primary cortical cultures, the blockade of N-methyl-D-aspartate (NMDA) glutamate receptors exacerbated neuronal apoptosis induced by serum deprivation. This effect is observed at early stage of cultures (9 days in vitro (DIV)) and mildly decreases in more mature cultures (13 and 15 DIV). At the opposite, low concentrations of NMDA (5 microM) or glutamate (5 microM) prevented the neuronal apoptosis induced by trophic support withdrawal. In primary cortical cultures, the proapoptotic effect of trophic support removal can be modulated by NMDA receptors depending upon the magnitude of these glutamate receptor activation.

Toxic neuronal apoptosis and modifications of tau and APP gene and protein expressions.

The causes and the mechanisms of neuronal death in Alzheimer's disease are not elucidated, although some new insights have been proposed over the past years, including free-radical toxicity, beta-amyloid toxicity, excitotoxicity, and disturbed cellular calcium metabolism. Some authors have also pointed out that apoptosis could play a role in neuronal degeneration, but it is still largely debated. Here, we review some recent data linking the induction of experimental neuronal apoptosis in vitro and the molecular pathology of the tau protein and amyloid precursor protein (APP). In cultures exposed to mild glutamate toxicity, tau mRNA expression, not beta-actin, is enhanced in stressed neurons. The Guam cycad toxin metabolite methylazoxymethanol also produces an increase of tau gene transcription that exacerbates changes induced by glutamate. In serum-deprived cultures or glutamate-exposed cultures, neurons committed to apoptosis have a reduced tau gene expression, whereas resistant neurons display a stable or even augmented tau mRNA expression accompanied by a persistent tau phosphorylation near serine 202. In the same conditions, stressed neurons produce membrane blebbings strongly immunopositive for APP and putative amyloidogenic fragments that are subsequently released in the extracellular space. Experimental apoptosis in neurons can recapitulate tau and APP modifications that could be associated with a selective vulnerability and a progression of cellular degeneration along the neuronal network.

The Guam cycad toxin methylazoxymethanol damages neuronal DNA and modulates tau mRNA expression and excitotoxicity.

As in Alzheimer's disease, brains of Guam Chamorros with amyotrophic lateral sclerosis (ALS) and Parkinsonism-dementia complex (PDC) contain intraneuronal-paired helical filaments composed of accumulated phosphorylated tau protein. Tau mRNA expression in rat neuronal cultures-normally modulated by glutamate-increases after treatment with the aglycone of cycasin, a cycad-derived toxin whose concentration in Chamorro food varies with disease incidence. Elevated Tau gene expression in vitro is coincident with increased cycasin-related DNA adducts and reduced DNA repair. Cycasin and endogenous glutamate may together promote the accumulation of tau protein and neuronal degeneration in Western Pacific ALS/PDC.

Mild kainate toxicity produces selective motoneuron death with marked activation of CA(2+)-permeable AMPA/kainate receptors.

Motoneuron death could be produced by higher sensitivity to excitoxicity during the development and pathological conditions. We report here that in ventral spinal cord cultures mild kainate exposure (12.5 microM, 20 min or 100 microM, 2.5 min) induced selective cobalt stain of motoneurons, indicating a specific Ca2+ entry through the Ca(2+)-permeable AMPA/kainate receptors. This result was associated with a selective motoneuron death as previously described. In these cultures, motoneuron immunoreactivity for the Ca2+ buffering protein, calretinin was negative. These findings suggest that the selective motoneuron death due to a mild excitotoxic insult could be linked to a marked Ca2+ influx associated with the lack of some Ca2+ buffering proteins.

Brefeldin A-induced apoptosis is expressed in rat neurons with dephosphorylated tau protein.

Brefeldin A (BFA) is a fungus metabolite (penicillum brefeldanum) that is known to produce the disintegration of the Golgi apparatus in exposed cells, and apoptosis in various cancer cells. This study reports that in rat primary cortical cell cultures BFA also produces apoptosis assessed by the TUNEL method and DAPI (4',6-diemidino-2-phenylindole) staining. The percentages of apoptotic neurons range from 26.9% +/- 8.3 to 43.2 +/- 2.5% in cultures exposed from 4 to 8 h to BFA (10 microg/ml). A double fluorescent staining, using AT8 antibody (phosphorylated tau) or tau1 antibody (dephosphorylated tau) associated with DAPI labeling reveals that tau1 positive neurons are more sensitive to BFA-induced apoptosis compared to AT8 positive neurons. This result and previous results using other apoptosis inducers suggest that tau phosphorylation in the vicinity of the AT8-tau1 epitopes is a marker of resistance or sensitivity to neuronal apoptosis in rat cortical cell cultures.

FK506 antagonizes apoptosis and c-jun protein expression in neuronal cultures.

FK506 is an immunosuppressive drug that binds to FK506 binding protein (FKBPs), a subgroup of cytosolic proteins called immunophillins. Previous works have revealed that FK506 protects neural cells from ischemia or excitotoxicity. Here we report that FK506 (10(-6) M) and not cyclosporine A (10(-6) M) blocks neuronal apoptosis induced by serum deprivation in rat neuronal cultures. In addition the immunohistochemical staining of C-jun protein in deprived cultures is markedly attenuated by FK506. The proportion of C-jun-positive neurons in control cultures, in serum-deprived cultures (48 h) and in serum-deprived cultures exposed to FK506 (10(-6) M) were 12.5%, 56.5% and 16.5%, respectively. The down-regulation of C-jun could play a major role in the anti-apoptotic action of FK506 in stressed neuronal cultures.

Neuronal apoptosis is associated with a decrease in tau mRNA expression.

Apoptosis is a programmed cell death that occurs during the development of the nervous system and in neurodegenerative disorders. Tau protein is a microtubule-associated protein which promotes microtubule polymerization and stabilization. Apoptosis was induced in primary neuronal cultures by glutamate exposure (200 microM, 15 min) or by serum deprivation, and tau mRNA levels were studied by quantitative in situ hybridization in apoptotic and non apoptotic neurons. Compared to controls, tau mRNA expression was decreased in apoptotic neurons produced by excitotoxicity or trophic support withdrawal. Under these conditions, resistant neurons to apoptosis display either increased tau mRNA levels after glutamate exposure or a stable tau mRNA expression after serum deprivation. In conclusion, in this in vitro model, neurons which are resistant and sensitive to apoptosis can be differentiated according to tau mRNA expression.

Urethral cytology and diagnosis of the pregnancy term. About some observations on the use of this method in an hospital setting.

A retrospective study of routine urethrocytograms performed over a 10-year period in 3,478 pregnant women for the diagnosis of pregnancy term was carried out to determine the reliability of urethral cytology when two, three, or four successive urethral smears were performed at 2- or 3-day intervals, and to investigate the correlations between the cytological results and the neurological ages of newborns. Microscopical examination of the 3,478 routine smears demonstrated the existence of a before-term aspect, a near-term aspect, or an at-term aspect in 7.4%, 66.7%, and 22.1% of cases, respectively. The diagnosis of post-term was noted only in 1 smear. Repeated urethrocytograms (1, 2, or 3 smears after the firstone) in women with a previously diagnosed before-term aspect or a near-term aspect revealed the occurence of the at-term aspect over time. Concordant correlations between cytological results and the neurological ages of the newborns were noted in 98.1% of cases. Three developmental stages (beginning, middle, end) of the near-term aspect and three stages of the at-term aspect were described. Due to its interest, the urethral-smear method should be worth generalizing as the other laboratory examinations that are routinely used to determine the term of pregnancy.

Glutamate toxicity enhances tau gene expression in neuronal cultures.

Tau protein is a microtubule-associated protein normally expressed in neurons. In Alzheimer's disease (AD) brains, phosphorylated tau accumulates in paired helical filaments which form neurofibrillary tangles in affected neurons; moreover, tau mRNA expression is increased in affected regions of AD brains. Glutamate, an excitatory neurotransmitter but also a potent neurotoxin under pathologic conditions, is known to produce neuronal degeneration and death accompanied by an increase in tau immunoreactivity in primary neuronal cultures. The goal of the present study is to evaluate the effects of glutamate on tau gene expression in neuronal cultures. We report a delayed and long-lasting enhancement of tau mRNA expression after a 15 min exposure to toxic concentrations of glutamate: neuronal tau mRNA levels reach a peak after 3 hr and remain increased 6 and 12 hr after the end of glutamate exposure. Both NMDA and AMPA/ kainate receptors are involved in this tau gene overexpression. Actinomycin D prevents this tau mRNA induction indicating that transduction signals elicited by glutamate act at the transcriptional level. The role of this delayed tau overexpression is not elucided but could be linked to either a reactive survival process or to a programmed cellular degeneration.

Cultured neurons expressing phosphorylated tau are more resistant to apoptosis induced by NMDA or serum deprivation.

Apoptosis is a programmed cell death that occurs during the development of the nervous system and in neurodegenerative disorders. Tau protein is a cytoskeletal component that promotes microtubule polymerization and stabilization. Apoptosis was induced in primary neuronal cultures by a prolonged exposure (16 h) to the NMDA (N-methyl-D-aspartate 20 microM) or by serum deprivation. The percentages of apoptotic neurons expressing phosphorylated tau (AT8) immunoreactivity are comparable in control and NMDA-exposed cultures (7.5 +/- 1.9 and 6.9 +/- 1.9%, respectively). At the opposite, the percentage of apoptotic neurons expressing de-phosphorylated tau (tau 1) immunolabelings is dramatically increased in NMDA-treated cultures (X 2.3 of controls). Similar results were also observed 48 h after serum deprivation. These results demonstrate in vitro that under these conditions, resistant and sensitive cortical neurons to apoptosis can be partly differentiated according to their phosphorylated tau immunoreactivities.

Increased hypoxic tolerance by chemical inhibition of oxidative phosphorylation: "chemical preconditioning".

A short ischemic episode preceding sustained ischemia is known to increase tolerance against ischemic cell death. We report early-onset long-lasting neuroprotection against in vitro hypoxia by preceding selective chemical inhibition of oxidative phosphorylation: "chemical preconditioning." The amplitude of CA1 population spikes (psap) in hippocampal slices prepared from control animals (control slices) was 31 +/- 27% (mean +/- SD) upon 45-min recovery from 15-min in vitro hypoxia. In slices prepared from animals treated in vivo with 20 mg/kg 3-nitropropionate (3-np) 1-24 h prior to slice preparation (preconditioned slices), psap improved to 90 +/- 15% (p < 0.01). Posthypoxic oxygen free radicals were reduced to 65 +/- 10% (mean +/- SD) of control in preconditioned slices (p < 0.05). Posthypoxic neuronal density improved from 52 +/- 15% (mean +/- SD) in control slices to 97 +/- 23% in preconditioned slices (p < 0.001). Glibenclamide, an antagonist at KATP-channels, partly reversed increased hypoxic tolerance. We conclude that chemical preconditioning induces early-onset long-lasting tolerance against in vitro hypoxia. Ultimately, this strategy may be applicable as a neuroprotective strategy in humans.