Alteration of bacterial adhesion induced by the substrate stiffness.

The aim of this paper is to study the impact of the substrate stiffness on the bacterial adhesion. For this purpose, agarose hydrogels are used as substrates with controlled mechanical properties. Indeed, the elastic modulus of these hydrogels, more precisely the shear storage moduli G', evolves with the agarose concentration (in this study from 0.75% to 3%). Other physico-chemical characteristics of the surface, known to be involved in bacterial adhesion, as hydrophobicity, were confirmed to remain constant. Two marine bacterial strains, a positive Gram Bacillus sp. 4J6 and a negative Gram Pseudoalteromonas sp. D41 were selected. Their retention on the substrates was analysed by confocal laser scanning microscopy and by counting of viable adhered bacteria. It was demonstrated that surface elastic modulus correlated with bacterial retention. Bacteria D41 adhered in higher numbers to rigid surfaces. For 4J6, bacterial adhesion patterns were changed: clusterings were observed on surfaces with lower elastic modulus. Furthermore, a proteomic study, based on the total soluble proteome of D41 strain, highlights an impact of elastic modulus on proteins synthesis. These data demonstrated an adapted response of adhering bacteria on hydrogels of varying mechanical properties.

Specific caspase pathways are activated in the two stages of cerebral infarction.

Necrosis and apoptosis have been initially identified as two exclusive pathways for cell death. In acute brain lesions, such as focal ischemia, this binary scheme is challenged by demonstrations of mixed morphological and biochemical characteristics of both apoptosis and necrosis in single cells. The resulting difficulty in defining the nature of cell death that is triggered by severe insults has dramatically impeded the development of therapeutic strategies. We show that in the early stages of cerebral infarction, neurons of the so-called "necrotic" core display a number of morphological, physiological, and biochemical features of early apoptosis, which include cytoplasmic and nuclear condensations and specific caspase activation cascades. Early activation cascades involve the death receptor pathway linked to caspase-8 and the caspase-1 pathway. They are not associated with alterations of mitochondrial respiration or activation of caspase-9. In contrast, pathways that are activated during the secondary expansion of the lesion in the penumbral area include caspase-9. In agreement with its downstream position in both mitochondria-dependent and -independent pathways, activation of caspase-3 displays a biphasic time course. We suggest that apoptosis is the first commitment to death after acute cerebral ischemia and that the final morphological features observed results from abortion of the process because of severe energy depletion in the core. In contrast, energy-dependent caspase activation cascades are observed in the penumbra in which apoptosis can fully develop because of residual blood supply.

Recruitment of the mitochondrial-dependent apoptotic pathway in amyotrophic lateral sclerosis.

Molecular mechanisms of apoptosis may participate in motor neuron degeneration produced by mutant superoxide dismutase-1 (mSOD1), the only proven cause of amyotrophic lateral sclerosis (ALS). Consistent with this, here we show that the proapoptotic protein Bax translocates from the cytosol to the mitochondria, whereas cytochrome c translocates from the mitochondria to the cytosol in spinal cords of transgenic mSOD1 mice during the progression of the disease. Concomitantly, caspase-9 is activated in the spinal cord of transgenic mSOD1 mice. Only in end-stage transgenic mSOD1 mice is the downstream caspase-7 activated and the inhibitor of apoptosis, XIAP, cleaved. These results indicate a sequential recruitment of molecular elements of the mitochondrial-dependent apoptotic pathway in transgenic mSOD1 mice. We also provide immunohistochemical evidence that cytochrome c translocation occurs in the spinal cord of sporadic ALS patients. Collectively, these data suggest that the mitochondrial-dependent apoptotic pathway may contribute to the demise of motor neurons in ALS and that targeting key molecules of this cascade may prove to be neuroprotective.

The role of glial cells in Parkinson's disease.

Parkinson's disease is a common neurodegenerative disorder characterized by the progressive loss of the dopaminergic neurons in the substantia nigra pars compacta. The loss of these neurons is associated with a glial response composed mainly of activated microglial cells and, to a lesser extent, of reactive astrocytes. This glial response may be the source of trophic factors and can protect against reactive oxygen species and glutamate. Aside from these beneficial effects, the glial response can mediate a variety of deleterious events related to the production of reactive species, and pro-inflammatory prostaglandin and cytokines. This article reviews the potential protective and deleterious effects of glial cells in the substantia nigra pars compacta of Parkinson's disease.

Increased expression of the pro-inflammatory enzyme cyclooxygenase-2 in amyotrophic lateral sclerosis.

Mutations in the copper/zinc superoxide dismutase (mSOD1) gene are associated with a familial form of amyotrophic lateral sclerosis (ALS), and their expression in transgenic mice produces an ALS-like syndrome. Recent observations suggest a role for inflammatory-related events in the progression and propagation of the neurodegenerative process in ALS. Consistent with this view, the present study demonstrates that, during the course of the disease, the expression of cyclooxygenase type 2 (Cox-2), a key enzyme in the synthesis of prostanoids, which are potent mediators of inflammation, is dramatically increased. In both early symptomatic and end-stage transgenic mSOD1 mice, neurons and, to a lesser extent, glial cells in the anterior horn of the spinal cord exhibit robust Cox-2 immunoreactivity. Cox-2 mRNA and protein levels and catalytic activity are also significantly increased in the spinal cord of the transgenic mSOD1 mice. The time course of the spinal cord Cox-2 upregulation parallels that of motor neuronal loss in transgenic mSOD1 mice. We also show that Cox-2 activity is dramatically increased in postmortem spinal cord samples from sporadic ALS patients. We speculate that Cox-2 upregulation, through its pivotal role in inflammation, is instrumental in the ALS neurodegenerative process and that Cox-2 inhibition may be a valuable therapeutic avenue for the treatment of ALS.

Analysis of 676 cases of ductal carcinoma in situ of the breast from 1971 to 1995: diagnosis and treatment--the experience of one institute.

Six hundred seventy-six patients with ductal carcinoma in situ of the breast (DCIS) from 1971 to 1995 were included in the study. Computerized patient files were retrospectively analyzed. Clinical findings were less frequently reported to reveal DCIS after 1989. Positive mammographic findings were obtained in 87% of patients and were mainly represented by microcalcifications (79.4%). Treatment procedures were breast-conserving surgery (BCS) alone (37.5%), BCS followed by radiation (BCSR) (25.5%), or mastectomy (M) (37%). The actuarial local recurrence was 2.6% in the M group (94 months of follow-up), 14.5% in the BCS group (85,7 months of follow-up), and 7.5% in the BCSR group (78.8 months of follow-up). Predictive factors of recurrence in all patients were invaded margin status and age. In the BCS group, grade was also a predictive factor. The analysis per decade shows that the lesions currently diagnosed are less serious than those of the past. All the recurrence in patients with positive margins was in the same quadrant as the original lesion. This further emphasizes the need for clear margins.

Developmental cell death in dopaminergic neurons of the substantia nigra of mice.

Dopaminergic neurons in the substantia nigra pars compacta (SNpc) undergo natural cell death during development in rats. Controversy exists as to the occurrence of this phenomenon in SNpc dopaminergic neurons in the developing mouse. Herein, by using an array of morphologic techniques, we show that many SNpc neurons fulfill the criteria for apoptosis and that the number of apoptotic neurons in the SNpc vary in a time-dependent manner from postnatal day 2 to 32. These dying neurons also show evidence of DNA fragmentation, of activated caspase-3, and of cleavage of beta-actin. Some, but not all of the SNpc apoptotic neurons still express their phenotypic marker tyrosine hydroxylase, confirming their dopaminergic nature. Consistent with the importance of target-derived trophic support in modulating developmental cell death, we demonstrate that destruction of intrinsic striatal neurons by a local injection of quinolinic acid (QA) dramatically enhances the magnitude of SNpc apoptosis and results in a lower number of adult SNpc dopaminergic neurons. Strengthening the apoptotic nature of the observed SNpc developmental cell death, we demonstrate that overexpression of the anti-apoptotic protein Bcl-2 attenuates both natural and QA-induced SNpc apoptosis. The present study provides compelling evidence that developmental neuronal death with a morphology of apoptosis does occur in the SNpc of mice and that this process plays a critical role in regulating the adult number of dopaminergic neurons in the SNpc.

Functional role of caspase-1 and caspase-3 in an ALS transgenic mouse model.

Mutations in the copper/zinc superoxide dismutase (SOD1) gene produce an animal model of familial amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder. To test a new therapeutic strategy for ALS, we examined the effect of caspase inhibition in transgenic mice expressing mutant human SOD1 with a substitution of glycine to alanine in position 93 (mSOD1(G93A)). Intracerebroventricular administration of zVAD-fmk, a broad caspase inhibitor, delays disease onset and mortality. Moreover, zVAD-fmk inhibits caspase-1 activity as well as caspase-1 and caspase-3 mRNA up-regulation, providing evidence for a non-cell-autonomous pathway regulating caspase expression. Caspases play an instrumental role in neurodegeneration in transgenic mSOD1(G93A) mice, which suggests that caspase inhibition may have a protective role in ALS.

Early and sequential recruitment of apoptotic effectors after focal permanent ischemia in mice.

In experimental models of cerebral ischemia, cells within the damaged territory die by necrosis and by apoptosis that contributes to the expansion of the insult. Apoptotic machinery mobilizes intracellular processes such as induction of Bcl-2 family members, activation of the proteolytic cascade including the caspases, and cleavage of caspase substrates, such as poly(ADP-ribose) polymerase or PARP. Mitochondria play a pivotal role in controlling apoptosis by releasing cytochrome c and modulating redox state, both under the regulation of manganese superoxide dismutase (Mn SOD) via superoxide anion detoxification. The implication and the kinetics of such events in apoptosis induced after focal permanent ischemia in mice remains to be studied. In a paradigm of ischemic insult induced by occlusion of the middle cerebral artery (MCAO) in mice, we showed by immunohistochemistry a constitutive expression of caspase-3 that is enhanced after MCAO in neurons localized within the infarcted zone. As a function of time intervals after MCAO, the cytochrome c amount increased in the cytosolic fraction of ischemic cortical extracts. The kinetics of the release was in concordance with the expression of caspase-3 and the subsequent cleavage of PARP appearing before the internucleosomal fragmentation of DNA, the ultimate step of apoptosis. When the apoptotic markers progressively appeared, no changes of Mn SOD activity or Mn SOD expression were detected after MCAO. We can therefore speculate that the recruitment of Mn SOD did not participate per se in the release of cytochrome c elicited after permanent focal ischemia.

Delaying caspase activation by Bcl-2: A clue to disease retardation in a transgenic mouse model of amyotrophic lateral sclerosis.

Molecular mechanisms of apoptosis may participate in motor neuron degeneration produced by mutant copper/zinc superoxide dismutase (mSOD1), the only proven cause of amyotrophic lateral sclerosis (ALS). Consistent with this, herein we show that the spinal cord of transgenic mSOD1 mice is the site of the sequential activation of caspase-1 and caspase-3. Activated caspase-3 and its produced beta-actin cleavage fragments are found in apoptotic neurons in the anterior horn of the spinal cord of affected transgenic mSOD1 mice; although such neurons are few, their scarcity should not undermine the potential importance of apoptosis in the overall mSOD1-related neurodegeneration. Overexpression of the anti-apoptotic protein Bcl-2 attenuates neurodegeneration and delays activation of the caspases and fragmentation of beta-actin. These data demonstrate that caspase activation occurs in this mouse model of ALS during neurodegeneration. Our study also suggests that modulation of caspase activity may provide protective benefit in the treatment of ALS, a view that is consistent with our recent demonstration of caspase inhibition extending the survival of transgenic mSOD1 mice.

Reduction of ischemic damage in NGF-transgenic mice: correlation with enhancement of antioxidant enzyme activities.

If permanent focal ischemia is induced by middle cerebral artery occlusion (MCAO), neurons within the infarcted territory die by necrosis and apoptosis (or programmed cell death). We have previously shown, using a mouse strain transgenic (tg) for the nerve growth factor (NGF) gene, that tg mice have consistently smaller infarcted areas than wild-type (wt) animals, correlated with upregulated NGF synthesis and impaired apoptotic cell death. We studied, in wt and tg mice subjected to MCAO, the activities of several antioxidant enzymes and the synthesis of the proteins of the Bcl-2 family. Our results show that the antiapoptotic Bcl-2 protein and glutathione peroxidase are recruited after MCAO. NGF-tg mice also had an intrinsic resistance to oxidative stress because their basal copper zinc superoxide dismutase (SOD) and glutathione transferase activities were high. Additionally, manganese SOD activity increased in NGF-tg mice after MCAO, correlating strongly with the resistance of these mice to apoptosis.

[Recurrence after treatment of breast intraductal carcinoma]. / Les récidives après traitement des carcinomes intracanalaires du sein.

OBJECTIVE: To assess the risk of local recurrence of intraductal carcinoma of the breast with a large series and a review of literature. METHODS: We present a retrospective study of 331 cases treated for intraductal carcinoma of the breast. Only patients with at least 5 years follow-up were selected. We were specially interested in recurrence risk factors. In these patients with a long follow-up; pathology was reevaluated with new investigation technique. RESULTS: After a median follow-up of 109 months, 40 local recurrences were observed; these lesions were invasive in 23 cases. Only one patient had recurrence after mastectomy. For the others, they had lumpectomy associated with radiotherapy in 12 cases. Histologic features, grade and therapeutic options were evaluated as risk factors of local recurrence. CONCLUSION: Follow-up after lumpectomy for intraductal carcinoma was studied. The status of tumor margins was important; irradiation appeared useful, specially in case of high grade carcinoma but further large prospective randomized studies are needed. The use of prognostic index is interesting, but there are still unanswered questions.

Reduction of cortical infarction and impairment of apoptosis in NGF-transgenic mice subjected to permanent focal ischemia.

The neuroprotective potential of the nerve growth factor (NGF) against permanent ischemic brain damage has been investigated in vivo using NGF-transgenic (tg) mice. The expression of the transgene is driven by part of the promoter of the proto-oncogene c-fos, which belongs to the first set of genes activated after brain ischemic insult. Wild-type (wt) mice and tg mice were subjected to permanent focal ischemia induced by electrocoagulation of the middle cerebral artery. Twenty four hours (h) after the ischemic shock, when compared to wt, tg mice displayed a 40% reduction of the infarcted area, which lasted up to 1 week. However, infarcted brain areas were similar in wt and tg mice within the first hours post-occlusion, indicating that NGF acted to block the progression of neuronal damage. Kinetics of NGF synthesis assessed by ELISA was in good agreement with the observed neuroprotective effect, since NGF content peaked 6 h post-ischemia. This was further correlated with the time-course of c-Fos immunoreactivity, detectable only from 6 h post-ischemia. The neuroprotective effect of NGF involved the impairment of apoptotic cell death, as evidenced by a marked decrease of the number of apoptotic profiles inside the ischemic zone in tg mice. These results underline the potential of c-fos-NGF-tg mice to study in vivo the molecular and cellular mechanisms of the NGF-induced neuroprotective effect against ischemic damage.

Recruitment of several neuroprotective pathways after permanent focal ischemia in mice.

After an ischemic episode induced by the electrocoagulation of the left middle cerebral artery (MCA) in mouse, neurons within the damaged territory die either by an apoptotic or by a necrotic process. Most of the cortical neurons within the ischemic area display both morphological and biochemical signs of programmed cell death: nuclear condensation, DNA degradation, formation of apoptotic bodies, and glutathione depletion. In fact, apoptosis essentially contributes to the expansion of the ischemic lesion and the maximum of damaged territory is reached 24 h postocclusion. Several potentially neuroprotective pathways have been evidenced in different experimental models of ischemia including the activation of antioxidant enzyme activities and/or the recruitment of neurotrophic as well as antiapoptotic factors. In our model of permanent focal ischemia induced by MCA occlusion, we measured the temporal synthesis of nerve growth factor (NGF) and examined the status of antioxidant enzymes as well as Bcl-2 antiapoptotic product. We detected in both cortices a transient increase of NGF which peaks at 6 h. Moreover, we reported that glutathione peroxidase is recruited with a time course which parallels NGF synthesis. Finally, we observed the induction of Bcl-2 in safe neurons; this may represent a self-protective response against ischemia-induced apoptosis. We provide evidence that in a model of permanent focal ischemia, several neuroprotective pathways could be coactivated.

c-Jun and cyclin D1 proteins as mediators of neuronal death after a focal ischaemic insult.

c-Jun, a transcriptional activator, as well as cyclin D1, a key regulator of the cell cycle, have been described in vitro as mediators of programmed neuronal death. After trophic factor deprivation, the activation of c-jun and cyclin D1 genes is considered as a necessary step within the cellular machinery that leads to cell death. We show here that both c-Jun and cyclin D1 proteins are present in neurones within the infarcted area after experimental cerebral ischaemia in the mouse. Since their presence was associated with DNA fragmentation revealed by the TUNEL procedure, we propose that c-Jun and cyclin D1 are involved in the process of neuronal death.

Apoptotic death in cortical neurons of mice subjected to focal ischemia.

Permanent focal cortical ischemia was induced in mice by electrocoagulation of the middle cerebral artery. At different time intervals after the injury, the volume of infarction was assessed together with an analysis of neuronal death. Morphological studies of ischemic brains and detection of nucleosomal DNA ladder within ipsilateral cortices might implicate a component of this neuronal loss to apoptosis as well as necrosis. Furthermore, we used the TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labelling) procedure to detect in situ DNA fragmentation. The localization and the proportion of apoptotic cells in the ischemic mouse brain would indicate that apoptosis contributes largely to the cellular loss induced by cerebral ischemia.

[Prenatal diagnosis and treatment of fetal hypothyroid goiter]. / Diagnostic et traitement anténataux d'un goitre foetal hypothyroïdien.

We report on a case of fetal goitrous hypothyroidism confirmed by the study of fetal thyroid function. Two injections of intra amniotic levo-thyroxine were performed at 35 and 36 WA. The serial ultra sonographic examination showed the disappearance of the fetal goiter. A healthy baby were delivered by cesarean section at 37 WA. At birth, the thyroid gland was slightly enlarged and the neonatal thyroid hormones were within the normal range. This case suggests that cordocentesis is a reliable method of assessing the status of the fetal thyroid, and that even as early as 35 WA a prenatal treatment of fetal goitrous hypothyroidism is possible by amniocentesis.